Transfection

Transfection
Recommended Promega Transfection Reagent for Commonly-Used Cell Lines.

Origin Human Human Human Human Human Monkey Monkey Mouse Hamster Hamster Rat Insect Cell Line HeLa Hep G2 293 K562 Jurkat COS-7 CV-1 NIH/3T3 BHK CHO PC12 Sf9 Cell Type Epithelial Hepatocyte Kidney transformed Lymphoblast T-cell leukemia Fibroblast Fibroblast Fibroblast Fibroblast Epitheliallike Pheochromocytoma Ovary TransFast Reagent Tfx-10 Reagent Tfx-20 Reagent Tfx-50 Reagent
X X X X X X X X X X X X X X
*Data were obtained from cells transiently transfected with plasmid DNA and using the Luciferase Assay System from Promega. Higher transfection efciencies were generally obtained with reagent:DNA complexes incubated with the cells in serum-free medium. Some cells exhibit similar transfection efciencies with several different reagents, thus more than one suggested reagent is indicated for these cell lines.
Preface
T R A N S F E C T I O N T O D E T E C T I O N . . .
Promega...
...the Source for Discovery
Promega Corporation is a worldwide leader in applying biochemistry and molecular biology to the development of innovative, high-value products for the life sciences. Products such as the Luciferase Assay Systema and the Dual-Luciferase Reporter Assay Systema,b have helped make Promega the leader in supplying genetic reporter systems for the study of eukaryotic gene expression and cellular physiology. Upstream from genetic reporting, Promegas family of transfection reagents are highly efcient, fast, easy to use and a tested complement to our reporter systems. Our commitment to your success in transfection and eukaryotic expression studies is reected in the detail found in this guide. The guide is complete with protocols, references and troubleshooting help, and features a cell line table on the inside cover to direct you to the Promega transfection reagent that will perform best with commonly-used cell lines. Please visit Promegas website at
Promegas Transfection, Reporter Assay Family and Eukaryotic Expression Vectors

Transfection Reagents
TransFast Transfection Reagentb Tfx-10 Reagentc Tfx-20 Reagentc Tfx-50 Reagentc Transfectam Reagentd for the Transfection of Eukaryotic Cells ProFection Mammalian Transfection Systems Calcium Phosphate DEAE-Dextran
Reporter Vectors and Assay Systems

Dual-Luciferase Reporter Assay Systema,b Luciferase Assay Systema CAT Enzyme Assay System -Galactosidase Enzyme Assay System Luciferase Reporter Vectors pGL3 Vectorse,f
Renilla Luciferase Control Vectors

pRL Vectorsg for the most current on-line references, applications, and other product information about our transfection reagents and reporter systems. Please also look for the Transfection Assistant to nd transfection conditions used successfully with Promega transfection reagents. Over 120 cell lines are represented. Promega truly is your source for transfection to detection reagents. CAT Reporter Vectors pSV--Galactosidase Control Vector
Eukaryotic Expression Vectors

pCI-neo Mammalian Expression Vectorh,i pCI Mammalian Expression Vectorh pSI Mammalian Expression Vector pTARGET Mammalian Expression Vector Systemh,j
aU.S.
Pat. Nos. 5,283,179, 5,641,641 and 5,650,289 have been issued to Promega Corporation for a rey luciferase assay method, which affords greater light output with improved kinetics as compared to the conventional assay. Pending.
bPatent cThe
cationic lipid component of the Tfx Reagents is covered by U.S. Patent No. 5,527,928 assigned to The Reagents of the University of California and pending foreign patents.
dTransfectam Reagent is covered by U.S. Patent No. 5,171,678. The Transfectam product was developed by J.P. Behr and J.P. Loefer (under license from CNRS-ULP Strasbourg). eU.S. fThe
Pat. No. 5,670,356 has been issued to Promega Corporation for a modied luciferase technology.
method of recombinant expression of Coleoptera luciferase is covered by U.S. Pat. No. 5,583,024 assigned to The Regents of the University of California.
gThe cDNA encoding luciferase from Renilla reniformis is covered by U.S. Pat. No. 5,292,658 assigned to the University of Georgia Research Foundation, Inc., and sublicensed from SeaLite Sciences, Inc., Norcross, GA. The pRL family of Renilla luciferase cDNA vectors is for research use only. hThe iU.S.
CMV vector technology is the subject of U.S. Pat. No. 5,168,062 assigned to the University of Iowa Research Foundation.
Pat. No. 4,766,072 has been issued to Promega Corporation for transcription vectors having two different bacteriophage RNA polymerase promoter sequences separated by a series of unique restriction sites into which foreign DNA can be inserted. under one or both of U.S. Pat. Nos. 5,487,993 and European Pat. No. 0 550 693.
jLicensed
Transfection Guide
Transfection Guide
Contents
T A B L E O F C O N T E N T S
Chapter 1
AN INTRODUCTION TO TRANSFECTION METHODS
Historical Background. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Transfection Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Chapter 2
P R E P A R A T I O N F O R T R A N S F E C T I O N
Preparation of DNA for Transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Preparation of Cells for Transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Transient Expression vs. Stable Transfection. . . . . . . . . . . . . . . . . . . . . . . 11
Chapter 3
CATIONIC LIPID TRANSFECTION REAGENTS
Cationic Lipid Transfection Reagents Transfectam, TransFast and Tfx Reagents for the Transfection of Eukaryotic Cells. . . . . . . . . . . . . . . . . . . 15 Liposome-Based Transfection Protocols TransFast and Tfx Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Transfection Protocols - Transfectam Reagent for the Transfection of Eukaryotic Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Optimization of Transfection Efciency. . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Chapter 4
PROFECTION MAMMALIAN TRANSFECTION SYSTEMS
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Calcium Phosphate-Mediated Transfection. . . . . . . . . . . . . . . . . . . . . . . . 29 Glycerol or DMSO Shock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 DEAE-Dextran-Mediated Transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Chapter 5
TROUBLESHOOTING TRANSFECTION REACTIONS
General Troubleshooting Tips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Cationic Lipid Reagent Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Calcium Phosphate Transfection Troubleshooting . . . . . . . . . . . . . . . . . . 36 DEAE-Dextran Transfection Troubleshooting . . . . . . . . . . . . . . . . . . . . . . 36
Chapter 6
G E N E T I C R E P O R T E R S Y S T E M S
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Firey Luciferase Reporter Gene Systems. . . . . . . . . . . . . . . . . . . . . . . . . 40 Dual-Luciferase Reporter Assay System. . . . . . . . . . . . . . . . . . . . . . . . . 42 CAT Reporter Gene Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 -Galactosidase Reporter Gene System . . . . . . . . . . . . . . . . . . . . . . . . . . 44 Mammalian Expression Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
pCAT, pGEM, PolyATtract, ProFection, RiboClone and Stop & Glo are trademarks of Promega Corporation and are registered with the U.S. Patent and Trademark Ofce. Dual-Luciferase, TransFast, pTARGET and Tfx are trademarks of Promega Corporation. Calbiochem is a registered trademark of CalbiochemNovabiochem Corporation. Corning is a trademark of Corning, Inc.
Cited References
R E F E R E N C E S
Fisherbrand is a registered trademark of Fisher Scientic. Geneticin is a registered trademark of Life Technologies, Inc. Kimwipes is a registered trademark of Kimberly Clark Corporation.
Cited References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Transfectam is a registered trademark of BioSepra, Inc. Transfectam product developed by J.P. Behr and J.P. Loefer (License of CNRS-ULP Strasbourg).
Appendix A
A D D I T I O N A L R E F E R E N C E S
References Using Promega Transfection Reagents. . . . . . . . . . . . . . . . . 49
Product claims are subject to change. Please contact Promega Technical Services or access the Promega on-line catalog for the most up-to-date information on Promega products. Applications mentioned in Promega literature are provided for informational purposes only. Promega does not warrant that referenced applications have been tested in Promega laboratories.
Appendix B
O R D E R I N G I N F O R M A T I O N
Ordering Information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
1998 Promega Corporation. All Rights Reserved.

Prices and specications subject to change without prior notice.
Transfection Guide
Transfection Guide
Chapter 1
Historical Background
The ability to introduce nucleic acids into cells has enabled the advancement of our knowledge of genetic regulation and protein function within eukaryotic cells, tissues and organisms. The successful pioneering studies of Vaheri and Pagano (1), and Graham and van der Eb (2) with DEAEdextran and calcium phosphate-mediated transfection techniques, paved the way for future experiments necessitating DNA transfer into cultured eukaryotic cells. The process of introducing nucleic acids into cells by non-viral methods, such as the DEAE-dextran and calcium phosphate techniques, is dened as transfection. This process is distinct from infection, which is a viral method of nucleic acid introduction into cells. Progress in transfection technology was relatively slow until the advent of molecular biology techniques for cloning plasmid DNA. These techniques provided the means to prepare and manipulate DNA sequences and the ability to prepare virtually unlimited amounts of relatively pure DNA for transfection experiments. Cloned sequences could also be used to generate RNA in vitro with phage RNA polymerase using DNA templates with the corresponding polymerase promoter (3). As the ability to prepare DNA and RNA for transfection became easier, additional methods, such as electroporation and liposome-mediated transfer, were developed to enable more efcient transfer of the nucleic acids to a broad range of cultured mammalian cells (4,5). The development of reporter gene systems and selection methods for stable gene expression of transferred DNA greatly expanded the applications for gene transfer technology (Figure 1.1). In 1982, Gorman et al. initiated the reporter gene concept
with the bacterial chloramphenicol acetyltransferase (CAT) gene and associated CAT assay system (6). Using a reporter gene that is not endogenous to the cell, coupled with a sensitive assay system for that gene product, allows investigators to clone regulatory sequences of interest upstream of the reporter gene to study expression of the reporter gene under various conditions. This technology, together with the availability of transfection reagents, provides the foundation for studying promoter and enhancer sequences, trans-acting proteins such as transcription factors, mRNA processing, protein/ protein interactions, translation, and recombination events (7). Since the introduction of the CAT gene and assay system several other reporter systems have been developed for various in vitro and in vivo applications including luciferase, -galactosidase, alkaline phosphatase and green uorescent protein (7). See Chapter 6 for detailed descriptions of Promegas luciferase, CAT and -galactosidase reporter vectors and assay systems. Integration of DNA into the chromosome, or stable episomal maintenance, of reporter genes and other genes occurs with a relatively low frequency. The ability to select for these cells is made possible using genes that encode resistance to a lethal drug. An example of such a combination is the marker gene for neomycin phosphotransferase with the drug Geneticin (8). Individual cells that survive the drug treatment expand into clonal groups that can be individually selected, propagated and analyzed. Today the study of gene regulation, the analysis of the expression and function of proteins within mammalian cells, the generation of transgenic organisms and in vivo/ex vivo gene therapy strategies are all made possible by the availability of of gene transfer technologies, nucleic acid molecular biology and reporter gene systems.
Reporter Gene Plasmid DNA
Nucleus
Reporter Protein
Cell Lysates Enzyme Activity
RNA
in situ -Galactosidase
Luciferase CAT -Galactosidase
Ribosomes
Figure 1.1. Reporter Gene Systems. Transfection Guide 5
Chapter 1
Transfection Technologies
Many transfection techniques have been developed. Desirable features include high efciency transfer of nucleic acid to the appropriate cellular organelle (for example, DNA into the nucleus), minimal intrusion or interference with normal cell physiology, low toxicity, ease of use, reproducibility, successful generation of stable transfectants, and in vivo efcacy. The techniques developed for gene transfer can be broadly classied as either chemical reagents or physical methods.
of nucleic acids into cells for transient expression; that is, for short-term expression studies of a few days in duration. However, this technique is not generally useful for stable transfection studies that rely upon integration of the transferred DNA into the chromosome (10). Other synthetic cationic polymers have been used for the transfer of DNA into cells, including polybrene (11), polyethyleneimine (12) and dendrimers (13,14). Calcium phosphate co-precipitation became a popular transfection technique following the systematic examination of this method by Graham and van der Eb in the now-classic paper published in 1972 (2). Their study examined the effect of different cations, cationic and phosphate concentrations, and pH on the parameters of transfection. Calcium phosphate co-precipitation is widely used because the components are easily available and reasonable in price, the protocol is easy to use and many different types of cultured cells can be transfected. This method is routinely used for both transient and stable transfection of a variety of cell types. The protocol involves mixing DNA with calcium chloride, adding this in a controlled manner to a buffered saline/phosphate solution and allowing the mixture to incubate at room temperature. This step generates a precipitate that
Chemical Reagents
DEAE-dextran was one of the rst chemical reagents used for transfer of nucleic acids into cultured mammalian cells (1,9). The ProFection Mammalian Transfection System-DEAE-Dextran provides reagents for this transfection technique (see Chapter 4 for further information). DEAE-dextran is a cationic polymer that associates with negatively charged nucleic acids. An excess of positive charge, contributed by the polymer in the DNA/polymer complex allows the complex to come into closer association with the negatively charged cell membrane. Uptake of the complex is presumably by endocytosis. This method is successful for delivery
+ Ca++

+ + +
+ +
+ +
Ca++ Ca++
Ca++ Ca++
Calcium Phosphate
DEAEDextran
Ca++ DNA DNA
+
+
+
+ +
+ +
+
+
+ +
+
+
+
+
+
+
DNA
Figure 1.2. Schematic representation of various transfection technologies based on chemical reagents.
Artificial Liposomes
Transfection Guide
Chapter 1
is dispersed onto the cultured cells. The precipitate is taken-up by the cells via endocytosis or phagocytosis. The calcium phosphate also appears to provide protection against intracellular and serum nucleases (15). Promegas ProFection Mammalian Transfection System-Calcium Phosphate provides reagents for this transfection technique (see Chapter 4 for further information). By 1980, articial liposomes were being used to deliver DNA into cells (5). The next advancement in liposomal vehicles was the development of synthetic cationic lipids by Felgner and colleagues (16). Liposome-mediated delivery offers advantages such as relatively high efciency of gene transfer, ability to transfect certain cell types that are intransigent to calcium phosphate or DEAE-dextran, successful delivery of DNA of all sizes from oligonucleotides to yeast articial chromosomes (16-20), delivery of RNA (21), and delivery of protein (22). Cells transfected by liposome techniques can be used for transient and for longer term experiments that rely upon integration of the DNA into the chromosome or episomal maintenance. Unlike the DEAE-dextran or calcium phosphate chemical methods, liposome-mediated nucleic acid delivery can be used for in vivo transfer of DNA and RNA to animals and humans (23). A lipid with overall net positive charge at physiological pH is the most common synthetic lipid component of liposomes developed for gene delivery (Figure 1.3). Often the cationic lipid is mixed with a neutral lipid such as L-dioleoyl phosphatidylethanolamine (DOPE) (Figure 1.4). The cationic portion of the lipid molecule associates with the negatively charged nucleic acids, resulting in compaction of the nucleic acid in a liposome/nucleic acid complex. For cultured cells, an overall net positive charge of the liposome/nucleic acid complex generally results in higher transfer efciencies, presumably because this allows closer association of the complex with the negatively charged cell membrane. Following endocytosis, the complexes appear in the endosomes, and later in the nucleus. It is unclear how the nucleic acids are released from the endosomes and traverse the nuclear membrane. DOPE is considered a fusogenic lipid (24) and it is thought that its role may be to release these complexes from the endosomes, as well as to facilitate fusion of the outer cell membrane with the liposome/nucleic acid complexes.
O
+
Promega provides a variety of transfection reagents that use cationic lipids for the delivery of nucleic acids to eukaryotic cells. These include TransFast Transfection Reagent, the Tfx Reagents and Transfectam Reagent. See Chapter 3 for more information on the use of these reagents.
Physical Methods
Direct microinjection into cultured cells or nuclei is an effective, although laborious technique to deliver nucleic acids into cells. This method has been used to transfer DNA into embryonic stem cells that are used to produce transgenic organisms (25). However, this technique is not appropriate for studies that require a large number of transfected cells. Electroporation was rst reported for gene transfer studies in 1982 (4). This technique is often used for cell types such as plant protoplasts that are particularly recalcitrant to milder methods of gene transfer. The mechanism for entry into the cell is based upon perturbation of the cell membrane by an electrical pulse, which forms pores that allow the passage of nucleic acids into the cell (26). The technique requires ne-tuning and optimization for duration and strength of the pulse for each type of cell used. A critical balance must be achieved between conditions that allow efcient delivery and conditions that kill cells. Another physical method of gene delivery is biolistic particle delivery. This method relies upon high velocity delivery of nucleic acids on microprojectiles to recipient cells (27). This method has been successfully employed to deliver nucleic acid to cultured cells, as well as to cells in vivo (28).
O O + N C O C O Cationic Head Group Link Lipid
Figure 1.3. General structure of a synthetic cationic lipid.
H3N
O O P OO
C O
Figure 1.4. Structure of DOPE (L-dioleoyl phosphatidylethanolamine).
Transfection Guide
Chapter 1
Notes
Transfection Guide
Chapter 2
Preparation of DNA for Transfection

The quality of the DNA used for transfection is critical. Puried plasmid DNA should be free from protein, RNA and chemical contamination. DNA may be puried using a plasmid preparation protocol, a CsCl gradient, or column chromatography. One measure of DNA purity is the ratio of absorbance at 260 to 280nm; for transfection the A260:A280 ratio should be at or above 1.8. The puried DNA should be ethanol precipitated and resuspended in sterile TE buffer to a nal concentration of approximately 1mg/ml. The optimal amount of DNA to use for transfection depends on both the cell type and the reagent used.
1. Harvest the bacterial cells from a 1 liter overnight culture by centrifugation at 6,000 x g for 10 minutes. If necessary, the cell pellet may be stored at 20C or 70C. 2. Resuspend the pellet in 50ml of 25mM Tris-HCl (pH 8.0), 50mM EDTA. 3. Add 100ml of freshly prepared 0.1M NaOH, 1% SDS; mix gently by swirling the container for ~15 seconds. Do not vortex. Incubate for 10 minutes on ice. 4. Add 75ml of ice-cold 5M potassium acetate. Mix gently and incubate on ice for 5 minutes. A precipitate will form. 5. Centrifuge at 6,000 x g for 15 minutes. Filter the supernatant through Miracloth or through 4 layers of cheesecloth. 6. Add 135ml of 2-propanol, mix and incubate at room temperature for 30 minutes. 7. Centrifuge at 6,000 x g for 15 minutes. Decant and discard the supernatant. 8. Resuspend the pellet in 20ml TE (pH 8.0). Add 20ml 5M ammonium acetate. Incubate on ice for 20 minutes. 9. Centrifuge at 12,000 x g for 10 minutes. Decant supernatant into a fresh tube. 10. Add 80ml of 100% ethanol to the supernatant. Incubate on ice for 15 minutes. Centrifuge at 12,000 x g for 10 minutes. 11. Dissolve pellet in 2ml TE (pH 8.0). Add 20l of 10mg/ml DNase-free RNase. Incubate for 15 minutes at 37C. 12. Add 600l 5M NaCl and 650l PEG precipitation solution. Mix and incubate on ice for 30 minutes. Centrifuge at 12,000 x g for 15 minutes at 4C. Discard the supernatant. Drain the pellets by inverting the tubes onto paper towels and blot the rim of the tube with a Kimwipes tissue or a paper towel. 13. Dissolve the pellet in 1ml TE (pH 8.0). Extract the remaining PEG by adding an equal volume of chloroform:isoamyl alcohol (24:1). Mix well by inversion and spin in a microcentrifuge for 5 minutes (or 1,600 x g for 10 minutes if using another rotor). 14. Remove the upper aqueous phase to fresh tubes. Add NaCl to a nal concentration of 0.5M (a total of 100l of 5M NaCl). Extract with an equal volume of high salt phenol. Spin for 5 minutes in a microcentrifuge tube. Remove the upper, aqueous phase to a fresh tube.
Plasmid Preparation Protocol

We have successfully puried transfection quality plasmid DNA using a modied alkaline lysis protocol (29). In the following procedure, membrane lipids are solubilized using SDS. Sodium hydroxide is used to denature and break up a large amount of the chromosomal DNA, which is then precipitated by addition of potassium acetate. Treatment with RNase A and ammonium acetate removes ribonucleic acids (30). Polyethylene glycol (PEG) is used to further purify the plasmid DNA by precipitating it away from other contaminating material (30). Any remaining proteins and oligosaccharides are removed by a high salt phenol extraction; the acid phenol extraction serves to remove residual chromosomal and nicked plasmid DNA. All reagents used should be molecular biology grade and solutions should be freshly prepared from reliable, nuclease-free stocks. Materials to Be Supplied by the User (Solution compositions are provided at the end of this chapter.) 25mM Tris-HCl, 50mM EDTA 0.1M NaOH, 1% SDS 5M potassium acetate TE (pH 8.0) 5M ammonium acetate 5M NaCl PEG precipitation solution DNase-free RNase chloroform:isoamyl alcohol (24:1) sterile water 2M sodium acetate high salt phenol acid phenol Miracloth (Calbiochem) 2-propanol 100% ethanol 70% ethanol sterile nuclease-free water
Transfection Guide
Chapter 2
15. Add two volumes of 100% ethanol. Incubate on ice for 15 minutes. Spin for 10 minutes in a microcentrifuge (20 minutes at 12,000 x g). Discard the supernatant and drain the pellets briey by inverting the tube onto paper towels. 16. Dissolve pellets in a total of 960l water. Add 15l of 5M NaCl and 25l of 2M sodium acetate (pH 4.0). Extract with an equal volume of acid phenol (31). Centrifuge for 5 minutes at room temperature in a microcentrifuge (phenol may crystallize at colder temperatures). 17. Extract any remaining phenol by adding an equal amount of chloroform:isoamyl alcohol (24:1). Invert to mix and centrifuge for 5 minutes in a microcentrifuge. Remove the upper aqueous phase to a fresh tube. 18. Add two volumes of 100% ethanol. Incubate for 20 minutes on ice or store overnight at 20C. Spin for 10 minutes in a microcentrifuge. Discard the supernatant. 19. Wash the pellet by adding 70% ethanol. Centrifuge for 10 minutes in a microcentrifuge. Carefully remove the supernatant without disturbing the pellet. Dry the pellet briey under vacuum. 20. Resuspend the DNA in 600l of sterile, nuclease-free TE. Determine the exact DNA concentration by measuring the absorbance at 260nm. Run an aliquot on a 0.7% agarose gel stained with ethidium bromide to check for the size, purity and integrity of the puried plasmid DNA. The above protocol is time consuming, but generates high quality DNA that works well in transfections. Alternatively, DNA puried by the alkaline lysis method may be further puried using a cesium chloride (CsCl) gradient.
Cesium Chloride Equilibrium Gradient

The CsCl equilibrium centrifugation method produces transfection quality DNA. Standard protocols for this procedure can be found in references 29 and 30. In this procedure, a highspeed centrifugation step follows a crude DNA isolation protocol such as the alkaline lysis procedure (29). Ethidium bromide (EtBr; a mutagen) is then added to the DNA along with CsCl and the mixture is centrifuged to equilibrium. The DNA band is removed, leaving many contaminants behind. A second EtBr and CsCl centrifugation removes additional protein and RNA contaminants. Once the DNA has been isolated, both EtBr and CsCl must be removed. EtBr may be removed using a Dowex AG50 column or by extraction with 1-butanol. The DNA must then be dialyzed or ethanol precipitated and washed thoroughly with 70% ethanol to remove excess CsCl. It is important to remove residual CsCl ions as they can react with charged liposomes or other transfection reagents, making transfection less effective. The puried DNA is resuspended in TE buffer.
Anion Exchange Chromatography

Column chromatography is by far the quickest method of preparing high quality plasmid DNA suitable for transfection. However, care must be taken in choosing the type of column used, as some commercially available columns leave contaminants in the DNA preparation that adversely affect transfection efciency, sometimes to a dramatic degree.
10
Transfection Guide
Chapter 2
Preparation of Cells for Transfection

Trypsinization Procedure for Removing Adherent Cells
Trypsinizing cells for purposes of subculturing or cell counting is an important technique that is critical to successful cell culture. The following technique works consistently well when passaging cells. Materials to Be Supplied by the User (Solution compositions are provided at the end of this chapter.) 1X trypsin-EDTA solution (0.05% trypsin, 0.5mM EDTA)
Transient Expression vs. Stable Transfection

Transient Expression
Cells are typically harvested 48-72 hours posttransfection for studies designed to analyze transient expression of the transfected genes. The optimal time interval depends upon the cell type, the doubling time of the cells and the specic characteristics of expression for the transferred gene. Analysis of gene products may require isolation of RNA or protein for enzymatic activity assays or immunoassays. The method used for cell harvest will depend upon the end-product being assayed. Extracts may be prepared using Promegas Reporter Lysis Buffer. This allows extracts to be assayed for luciferase, CAT and -galactosidase activity. If only luciferase activity is to be assayed, Promegas Cell Culture Lysis Reagent may be used. Passive Lysis Buffer is best for the Dual-Luciferase Reporter Assay System. For further information on the preparation and assay of cell extracts, see Chapter 6.
1. Prepare a sterile trypsin-EDTA solution in a calcium- and magnesium-free salt solution such as 1X PBS or 1X HBSS. The 1X solution can be frozen and thawed for future use, but the activity of the trypsin will decline with each freeze-thaw cycle. The trypsin-EDTA solution may be stored for up to 1 month at 4C. 2. Remove the media from the tissue culture dish. Add enough PBS or HBS solution to cover the cell monolayer: 2ml for a 150mm ask, 1ml for a 100mm plate. Rock the plates to distribute the solution evenly. Remove and repeat the wash. Remove the nal wash. Add enough trypsin solution to cover the cell monolayer. 3. Place the plates in a 37C incubator until the cells just begin to detach (usually 1-2 minutes). 4. Remove the ask from the incubator. Strike the bottom and sides of the culture vessel sharply with the palm of your hand to help dislodge the remaining adherent cells. View the cells under a microscope to check whether all cells have detached from the growth surface. If necessary, the cells may be returned to the incubator for an additional 1-2 minutes. 5. When all cells have detached, add media containing serum to the cells to inactivate the trypsin. Gently pipet the cells up and down to break up cell clumps. The cells may then be counted using a hemacytometer and/or distributed to fresh plates for subculturing.
Stable Transfection
The goal of stable, long-term transfection is to isolate and propagate individual clones containing transfected DNA. Therefore it is necessary to distinguish nontransfected cells from those that have taken up the exogenous DNA. This screening can be accomplished by drug selection when an appropriate drug resistance marker is included in the transfected DNA. Alternatively, morphological transformation can be used as a selectable trait in certain cases. For example, bovine papilloma virus vectors produce a morphological change in transfected mouse CI127 cells (32). Typically, cells are maintained in nonselective medium for 1-2 days post-transfection, then trypsinized and replated in selective medium containing the drug. The use of the selective medium is continued for 2-3 weeks, with frequent changes of medium to eliminate dead cells and debris, until distinct colonies can be visualized. Individual colonies are then trypsinized and subcloned to multiwell plates for further propagation in the presence of selective medium.
Transfection Guide
11
Chapter 2
Several different drug selection markers are commonly used for long-term transfection studies. For example, cells transfected with recombinant vectors containing the bacterial gene for neomycin phosphotransferase can be selected for stable transformation in the presence of the neomycin analog G418 (8). Similarly, expression of the gene for hygromycin B phosphotransferase from the transfected vector will confer resistance to the drug hygromycin B (33). An alternative strategy is to use a vector carrying an essential gene that is defective in a given cell line. For example, CHO cells decient in expression of the dihydrofolate reductase (DHFR) gene survive only in the presence of added nucleosides.
However, these cells, when stably transfected with DNA expressing the DHFR gene, will synthesize the required nucleosides (34). An additional advantage of using DHFR as a marker is that gene amplication of DHFR and associated transfected DNA occurs when cells are exposed to increasing doses of methotrexate (35). Before using a particular drug for selection purposes, it is important to determine the amount of drug necessary to kill the cells you will be using. This may vary greatly between cell types. Design experiments using various concentrations of the drug to determine the amount to use for selection of resistant clones (29).
Transient Transfection
DNA sample
Assay for Gene Expression
Day 1
Day 3 or 4
Stable Transfection
DNA sample
Apply Selective Pressure
Select clonal cells that stably replicate and express transfected DNA.
Day 1
Day 2 or 3
2-3 Weeks
Figure 2.1. Schematic representations of stable and transient transfections.
12
Transfection Guide
Chapter 2
Composition of Buffers and Solutions

5M ammonium acetate Dissolve 385g of ammonium acetate in 1 liter distilled water. Filter through a 0.2m lter. Store at 4C. DNase-free RNase A Prepare a 10mg/ml stock solution of Pancreatic RNase A in 10mM Tris-HCl (pH 7.5), 15mM NaCl. Aliquot to tubes and heat in a boiling water bath for 15 minutes. Cool slowly to room temperature. Store at 20C. 1X HBSS (Hanks Balanced Salt Solution) 5mM KCl 0.3mM KH2PO4 138mM NaCl 4mM NaHCO3 0.3mM Na2HPO4 5.6mM D-glucose The nal pH should be 7.1 1X PBS 137mM 2.7mM 4.3mM 1.47mM
High Salt Phenol (phenol saturated with TE + 0.5M NaCl) Phenol is caustic; work in a chemical safety hood and wear protective safety equipment. Melt phenol by placing in a 50C or warmer water bath. Add an equal volume of TES (TE + 1/10 volume of 5M NaCl). Stir with a Teon-coated magnetic stir bar until the two phases become completely mixed. Stop stirring and allow the phases to separate. Remove and discard the top aqueous phase. Add TES, mix, and allow the phases to separate; remove the aqueous phase. Repeat two more times. Add back 1/10 volume of TES to the phenol. Store protected from light at 4C. 5M Potassium Acetate Solution 60ml 5M potassium acetate 11.5ml glacial acetic acid 28.5ml deionized water Store at 4C. This solution is 3M with respect to potassium and 5M with respect to acetate. 2M Sodium Acetate, pH 4.0 15g NaOH 115ml deionized water 115ml glacial acetic acid Dissolve the NaOH slowly in 115ml of water. Slowly add the glacial acetic acid. Adjust the nal volume to 1L with deionized water. The nal pH of the solution should be 4.0. This solution provides a 40X stock for the 50mM sodium acetate solution used to prepare acid phenol. TE 10mM 1mM Tris-HCl (pH 8.0) EDTA
NaCl KCl Na2HPO4 KH2PO4
The nal pH should be 7.1 PEG Precipitation Solution (30% PEG-8000, 1.5M NaCl) 300g PEG 8000 (molecular biology grade) 300ml 5M NaCl Add deionized water to a nal volume of 1L. This solution may have to be heated slightly to completely dissolve the PEG. Store at 4C. Acid Phenol (phenol saturated with TE + 50mM sodium acetate) Phenol is caustic; work in a chemical safety hood and wear protective safety equipment. Melt phenol by placing in a 50C or warmer water bath. Add an equal volume of 50mM sodium acetate (pH 4.0). The pH of the sodium acetate solution is important. Stir with a Teon-coated magnetic stir bar until the two phases become completely mixed. Stop stirring and allow the phases to separate. Remove and discard the top aqueous phase. Add 50mM sodium acetate (pH 4.0), mix and allow the phases to separate. Remove the aqueous phase. Repeat two more times or until the pH of the aqueous phase after extraction is between 4.0 and 4.2. Add back 1/10 volume of 50mM sodium acetate (pH 4.0) to the phenol. Store protected from light at 4C.
1X Trypsin-EDTA solution 0.05% (w/v) trypsin 0.53mM EDTA Dissolve in a calcium- and magnesium-free salt solution such as 1X PBS or 1X HBSS.
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Chapter 2
Notes
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Cationic Lipid Transfection Reagents Transfectam, TransFast and Tfx Reagents for the Transfection of Eukaryotic Cells
Introduction to Promegas Cationic Lipid Reagents
Promega provides three types of cationic lipidbased transfection reagents, Transfectam Reagent, TransFast Reagent and the Tfx-10, Tfx-20 and Tfx-50 Reagents. The cationic lipid component of these reagents associates with negatively charged nucleic acids, resulting in a lipid/nucleic acid complex that has a net neutral or positive charge and therefore allows closer association of the DNA with the negatively charged cell membrane. Transfectam Reagent for the Transfection of Eukaryotic Cells is a cationic lipid reagent consisting of dioctadecylamidoglycyl spermine (DOGS), a synthetic, cationic lipopolyamine. The spermine group is covalently attached through a peptide bond to the lipid moiety (Figure 3.1). The strong positive charge contributed by the spermine headgroup gives the molecule a high afnity for DNA (105106M 1). The positively charged headgroup effectively coats the negatively charged DNA with a cationic lipid layer, allowing it to fuse with the plasma membrane of eukaryotic cells, resulting in internalization of the DNA.
Incubation of cationic lipid-containing liposomes and nucleic acids results in quick association and a compaction of the nucleic acid (39,40), presumably from electrostatic interactions between the negatively charged nucleic acid and the positively charged head group of the synthetic lipid. Entry of the liposome complex into the cell may occur by the processes of endocytosis, or fusion with the plasma membrane via the lipid moieties of the liposome (41). Once inside the cell, the complexes often become trapped in endosomes and lysosomes. Endosomal disruption is facilitated by DOPE (24), which allows the complexes to escape into the cytoplasm. The cytoplasm is the site of action for RNA or anti-sense oligonucleotides delivered via the liposomes. The nucleus is the target for most DNA delivery and it is not known precisely how the transfected DNA or liposome/DNA complex gains entry to the nucleus. Promegas TransFast and Tfx Reagents facilitate liposome-mediated transfer of nucleic acids into eukaryotic cells. The TransFast Reagent is composed of the synthetic cationic lipid, N,N [bis (2hydroxyethyl)]-N-methyl-N-[2,3 di(tetradecanoyloxy) propyl] ammonium iodide (Figure 3.2a) and the neutral lipid, (DOPE) (Figure 1.4). The Tfx Reagents contain a mixture of a synthetic, cationic lipid molecule [N,N,N,N-tetramethyl-N,Nbis)2-hydroxy-ethyl)-2,3,-dioleoyloxy-1,4butanediammonium iodide] (Figure 3.2b) and DOPE (Figure 1.4). All of the Tfx Reagents (Tfx-10, Tfx20, and Tfx-50) contain the same concentration of the cationic lipid component, but contain different molar ratios of the fusogenic lipid, DOPE. The best transfection reagent and conditions for a particular cell type must be empirically and systematically tested because inherent properties of the cell inuence the success of any specic transfection method.
CH3 N HO I
+
+NH
+NH
O N C
NH C +NH 2 O
O O O O
+NH
HO
Figure 3.1. Structure of Transfectam Reagent.
Liposome Based Transfection Reagents

The term liposome refers to lipid bilayers that form colloidal particles in an aqueous medium (36). Liposome reagents specically designed for transfection applications incorporate synthetic cationic lipids (16), often formulated together with the neutral lipid DOPE (Figure 1.4), which has been demonstrated to enhance the gene transfer ability of certain synthetic cationic lipids (37,38).
Figure 3.2a. Structure of the synthetic cationic lipid component of the TransFast Reagent.
HO N
I O C
HO
N+
C O
Figure 3.2b. Structure of the synthetic cationic lipid component of the Tfx Reagents.
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Advantages of Using Cationic Lipid Reagents for Transfection

Cationic lipid reagents designed for transfection applications are more versatile than many other traditional methods. The advantages include versatility in the macromolecules delivered, in vitro and in vivo applications, ability to reproducibly transfect cells that are recalcitrant to other methods, and suitability for transient and stable transfection paradigms. For example, several different types of macromolecules can be delivered to cells using these methods, including RNA and DNA of all sizes ranging from oligonucleotides to plasmids and yeast articial chromosomes (17-21,42). TransFast Reagent, Transfectam Reagent and the Tfx Reagents offer the advantages that they are easy to optimize and work well for a variety of cell types (43-46). In addition, these reagents are excellent for use with primary cells as they can be used in the presence of serum, can be used for both transient and stable transfections and are of low toxicity.
A. CHO Cells
4:1 Ratio 25 2:1 Ratio
The Tfx and Transfectam Reagents can also be used for in vivo transfection (47-49). It has been shown that Tfx-50 Reagent is highly active in the presence of amniotic uid (50), which has implications for its use in intra-amniotic injection and transfection.
Factors Inuencing Transfection Efciency

With any transfection reagent or method, cell health, degree of conuency, passage number, contamination, and DNA quality and quantity are important parameters that can greatly inuence transfection efciency. Plasmid DNA for transfections should be free of protein, RNA and chemical contamination (See Chapter 2). Suspend ethanol-precipitated DNA in sterile water or TE buffer to a nal concentration of 0.2-1mg/ml. The optimal amount of DNA to use in the transfection will vary widely depending upon the type of DNA and the target cell line. It is essential to optimize specic transfection conditions to gain optimal transfection efciencies.
B. HeLa Cells
4:1 Ratio 50 45 40 35 30 25 20 15 10 5 0 125 250 500 1,000 125 250 500 1,000 250 2:1 Ratio
Relative Light Units (x 10-4)
20 15
10 5 0 125 250 500 1,000 125 250 500 1,000 1,000
ng DNA Tfx-10 Tfx-20 Tfx-50 Tfx-10
ng DNA Tfx-20 Tfx-50
C. BHK Cells
4:1 Ratio 180 2:1 Ratio 45
D. 293 Cells
4:1 Ratio 2:1 Ratio
140 120 100 80 60 40 20 0 125 250 500 1,000 125 250 500 1,000 500
160
40 35 30 25 20 15 10 5 0 125 250 500 1,000 125 250 500 1,000 1,000
ng DNA Tfx-10 Tfx-20 Tfx-50 Tfx-10
ng DNA Tfx-20 Tfx-50
Figure 3.3. Relative levels of gene expression as a function of Tfx Reagent, DNA amount and reagent:DNA charge ratio. CHO Cells (Panel A), HeLa cells (Panel B), BHK cells (Panel C) and 293 cells (Panel D) were plated at a density of 50,000 cells/well in 24 well plates. Transfections were performed in the absence of serum using the indicated Tfx Reagent and pGL3-Control Vector at reagent:DNA ratios of 2:1 and 4:1. All transfections were overlaid with serum-containing media after one hour, and cells were harvested for luciferase assays after 48 hours. The results represent the mean of 6 replicates and are expressed as relative light units per well of cells. The single Tfx-50 Reagent conditions reect the optimal DNA amount and reagent:DNA ratio determined from previous optimization experiments. 16 Transfection Guide
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The important parameters to optimize in order to maximize transfection efciencies are the charge ratio of transfection reagent to DNA, the amount of transfected DNA, the length of time the cells are exposed to the transfection reagent and the presence or absence of serum. Figure 3.3 shows an example of optimization experiments for 4 different cell lines using different amounts of pGL3 Control DNA, different reagent:DNA charge ratios and the three different Tfx Reagents. Expression of luciferase activity from the transfected DNA is indicated in relative light units on the Y-axis. The graphs show the results of comparisons among the three Tfx Reagents: for CHO cells, Tfx-10 Reagent and 500ng DNA at a 2:1 reagent:DNA charge ratio was most effective; for HeLa cells, Tfx-20 Reagent and 250ng DNA at a 2:1 charge ratio was most effective; for BHK cells Tfx-10 Reagent and 1,000ng DNA at a 2:1 ratio was most effective; and for 293 cells Tfx-20 Reagent and 500ng of DNA at a 2:1 Reagent:DNA ratio was most effective. It should be noted that Figure 3.3 is a comparison of the performance of the three Tfx Reagents in these cell lines. For CHO and 293 cells, TransFast Reagent has been found to perform better than Tfx Reagents. The transfection efciency achieved using all of Promegas cationic lipid-based transfection reagents varies depending on the cell type being transfected and the transfection conditions used.
DNA The optimal amount of DNA to use in the transfection will vary depending upon the type of DNA and the target cell line used. For example, HeLa cells are optimally transfected with 0.25g of pGL3-Control DNA using Tfx-20 Reagent while NIH/3T3 cells are optimally transfected with TransFast Reagent. For adherent cells, we recommend initially testing 0.25, 0.50, 0.75 and 1g of DNA in a 24 well plate format at a transfection reagent:DNA ratio of 3:1 for each of the Tfx Reagents and at transfection reagent:DNA ratios of 2:1 and 1:1 for TransFast Reagent. Increasing the amount of DNA does not necessarily result in higher transfection efciencies. Time The optimal transfection time is dependent upon the cell line and DNA used. For the rst tests, use a one hour transfection interval. However, in optimization experiments, test transfection times from 30 minutes to 4 hours. Monitor cell morphology during the transfection interval, particularly when the cells are maintained in serum-free medium, as some cell lines lose viability under these conditions. The transfection time with the TransFast and Tfx Reagents is usually signicantly shorter than that required with other cationic lipid compounds, and can be decreased to as little as 30 minutes with certain cell lines (Figure 3.4). In addition to saving time, this shortened transfection time may signicantly reduce the risk of cell death during the transfection procedure.
Liposome Based Transfection Protocols TransFast and Tfx Reagents

General Considerations
Charge Ratio of Transfection Reagent to DNA The amount of positive charge contributed by the cationic lipid component of the transfection reagent should equal or exceed the amount of negative charge contributed by the phosphates on the DNA backbone, resulting in a net neutral or positive charge on the multilamellar vesicles associating with the DNA. Charge ratios of 2:1 to 4:1 Tfx Reagent:DNA and 1:1 to 2:1 TransFast Reagent:DNA have worked well with various cultured cells but ratios outside of this range may be optimal for other cell types or applications. Each of the Tfx Reagents contains the same amount of cationic lipid (1mM when the contents of each vial are resuspended in the recommended 400l volume), but contains varying amounts of the neutral lipid component, DOPE.
Relative Light Units
300,000
250,000
200,000
150,000
100,000
50,000
30 Minutes
1 Hour
2 Hours
4 Hours
Transfection Interval
Figure 3.4. Effect of transfection interval on transfection of CHO cells using TransFast Reagent. CHO cells were transfected with 250ng of pGL3-Control DNA using TransFast Reagent at a 2:1 reagent: DNA charge ratio for various times in the absence of serum. All transfections were performed in 24 well plates and cell lysates were harvested 2 days post-transfection. The results represent the mean of 6 replicates and are expressed as relative light units per well.
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Serum Transfection protocols often require serum-free conditions for optimal performance, as serum can interfere with many commercially available transfection reagents. The TransFast and Tfx Reagents can be used in transfection protocols in the presence of serum, allowing transfection of cell types or applications that require continuous exposure to serum. Figure 3.5 shows the effect of the presence or absence or serum on transfection of COS-7 cells using TransFast Reagent.
Stable Transfection The TransFast and Tfx Reagents can be used for the production of stable transfectants. However, we recommend rst optimizing the transfection conditions using transient transfection studies. Lipid Carrier The TransFast and each of the Tfx Reagents work optimally for different cell lines. For example, we have determined that BHK cells are optimally transfected with Tfx-10 Reagent, HeLa cells with Tfx-20 Reagent, and 293 cells with TransFast Reagent (see the table on the inside front cover of this guide). The optimal transfection reagent for each cell line needs to be determined empirically. Table 3.1 gives specic transfection conditions that have worked well for the various Tfx Reagents and TransFast Reagent in some commonly-used cell lines.
250,000
Relative Light Units/Well
200,000 150,000 100,000 50,000
+ Serum
Serum
Figure 3.5. Effect of serum on transfection of COS-7 cells using TransFast Reagent. Cells were transfected with 500ng of a CMVpromoter driven luciferase reporter plasmid DNA per well, at 1:1 reagent:DNA charge ratios in 10% serum-supplemented or serum-free medium. The transfection interval was one hour. All transfections were performed in 24 well plates and cell lysates were harvested 2 days post-transfection. The results represent the mean of 6 replicates and are expressed as relative light units per well.
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Table 3.1. A Comparison of Transfection Conditions Used for TransFast, Tfx-10, Tfx-20 and Tfx-50 Reagents with Various Cell Lines. Transfection Solution per Well in a 24 Well Plate 250ng plasmid DNA 0.75l TransFast Reagent S.F. Media to 200l 1.0g plasmid DNA 3.0l Tfx-10 S.F. Media to 200l 500ng plasmid DNA 1.5l TransFast Reagent S.F. Media to 200l 1.0g plasmid DNA 3l TransFast Reagent Serum + Media to 200l 500ng plasmid DNA 1.5l TransFast Reagent Serum + Media to 200l (or S.F. Media to 200l) 1.0g plasmid DNA 3l TransFast Reagent S.F. Media to 200l 250ng plasmid DNA 0.75l Tfx-20 S.F. Media to 200l 250ng plasmid DNA 1.5l Tfx-20 S.F. Media to 200l 250ng plasmid DNA 1.1l Tfx-50 (or 1.5l) Serum + Media to 200l 3g plasmid DNA 9l TransFast Reagent S.F. Media to 1ml per 6 well plate 4g plasmid DNA 12l (or 24l) TransFast Reagent S.F. Media to 1ml per 6 well plate 1.0g plasmid DNA 3.0l TransFast Reagent S.F. Media to 200l 1.0g plasmid DNA 2.25l Tfx-20 S.F. Media to 200l 1.0g plasmid DNA 6l TransFast Reagent S.F. Media to 200l 500ng plasmid DNA 1.5l Tfx-20 Reagent S.F. Media to 200l 500ng plasmid DNA 3l TransFast Reagent S.F. Media to 200l Charge Ratio 1:1
Cell Line 293
Cell Type Attached; Human Epithelial; Ad5-transformed embryonic kidney Attached; Hamster Fibroblasts; Kidney Attached; Hamster Epithelial-like; Ovary
Reagent TransFast Reagent
BHK
Tfx-10
2:1
CHO
TransFast Reagent
1:1 (2:1)
TransFast Reagent COS-7 Attached; Monkey Fibroblasts; African Green Monkey Kidney; SV40 Transformed Attached; Monkey Fibroblasts; African Green Monkey Kidney Attached; Human Epithelial; cervical carcinoma Attached; Human Epithelial; Hepatoblastoma TransFast Reagent
1:1
1:1
CV-1
TransFast Reagent
1:1
HeLa
Tfx-20
2:1
HepG2 1
Tfx-20
4:1
Tfx-50
3:1 (4:1) 1:1
Jurkat 2
Suspension; Human T-lymphocytes; T cell leukemia Suspension; Human Lymphoblast; Myelogenous Leukemia Attached; Mouse Fibroblasts; NIH Swiss Mouse embryo Attached; Rat Adrenal; Pheochromocytoma
TransFast Reagent TransFast Reagent TransFast Reagent Tfx-20
K562 2
1:1 (2:1) 1:1
NIH/3T3
PC12
1.5:1
TransFast Reagent SF9 Insect Tfx-20
2:1
2:1
TransFast Reagent
N.D. = Not Determined S.F. = Serum-Free
1TransFast 2Procedures
2:1
Reagent Not Tested. are different for suspension cells. See page 23.
Note: Conditions for these cell lines were determined using cells obtained from the American Type Culture Collection (ATCC). All attached cells were tested at low passage number and 80% conuency.
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Protocol A general protocol for use with Promegas liposomebased transfection reagents (TransFast, Tfx-10, Tfx-20 and Tfx-50 Reagents) is provided below. Figure 3.6 gives a general overview of the steps involved in the procedure. This protocol can be used with serum-supplemented or serum-free medium. For further information on each system, please request the TransFast Transfection Reagent Technical Bulletin #TB260, or the Tfx Reagents Technical Bulletin #TB216. These Technical Bulletins are also available on the Internet at www.promega.com. For a list of references using the Tfx Reagents in a variety of cell lines, see Appendix A.
Materials to Be Supplied by the User cell culture medium with serum (i.e., complete medium; appropriate for the cell type being transfected) serum-free cell culture medium 24 well plates or 60mm or 100mm cell culture plates
Liposomes
Day One Plate cells, resuspend and freeze liposome reagent.
1. Add liposome reagent to the DNA/Medium mixture and vortex briefly.
Day Two Dilute DNA in medium.
Media + DNA
Add thawed liposome reagent to the DNA /medium mixture and vortex briefly.
Incubate the DNA/liposome reagent mixture for 10-15 minutes at room temperature.
2. Incubate 10-15 minutes.
Remove the growth medium from the cells.

DNA/ Liposome Complex
Add the transfection mixture to the cells and return them to the 37C incubator.
After an incubation period (usually 1 hour), add complete growth medium to cells.
Return the cells to the incubator for the appropriate length of time before analysis.
3. Add DNA/ liposome complex to cells.
Perform the desired assay.
Figure 3.6. Overview of cationic lipid - mediated transfection with adherant cells.
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Plating Cells
Cells should be almost conuent by the time they are harvested 48 hours after transfection. The degree of conuency on the day of transfection is a parameter that needs to be optimized for each individual cell line. As a general guideline, plate cells one day before the transfection experiment so that they will be approximately 50-80% conuent on the day of the transfection. Some cell lines, such as HeLa cells, exhibit higher toxicity effects when transfected at lower cell densities. As a general guideline, plate 5 x 104 cells per well (24 well plate) or 5.5 x 105 cells (60mm culture dish). Change cell numbers proportionately for differently sized plates (see Table 3.2).
Table 3.2. Area of Culture Plates for Cell Growth. Growth Area (cm2)a 1.88 0.32 3.83 9.4 8.0 21 55
2. Before each use, thaw at room temperature and vortex the solution. If liquid has condensed at the top of the vial or in the vial cap, collect the liquid by placing the reagent vial inside a 50ml centrifuge tube and centrifuging briey at 300 x g. After use, store the remaining stock in the vial at 20C.
Optimization of Transfection
Plasmids with reporter gene functions can be used to monitor transfection efciencies (see Chapter 6). An ideal reporter gene product is one that is unique to the cell, can be expressed from plasmid DNA and can be assayed conveniently. Generally, reporter gene assays are performed 2-3 days after transfection. We recommend testing various amounts of transfected DNA (0.25, 0.5, 0.75 and 1.0g per well), using a one-hour exposure time and charge ratios of TransFast Reagent:DNA of 1:1 and 2:1, or a 3:1 ratio of Tfx Reagent:DNA. This can be done under serum-free conditions with adherent cells in a 24 well plate format. Figure 3.7 outlines a typical optimization matrix. 1:1 Charge Ratio of TransFast Reagent:DNA
g DNA/well 0.25 0.50 0.75 1.0
Size of Plate 24 well 96 well 12 well 6 well 35mm 60mm 100mm

a
Relative Areab 1X 0.2 X 2X 5X 4.2 X 11 X 29 X
This information was calculated for Corning culture dishes.
Relative area is expressed as a factor of the total growth area of the 24 well plate recommended for optimization studies. To determine the proper plating density, multiply 5 x 104 cells by this factor.
Preparation of Liposome Reagent Stock Solution

1. The day before transfection, warm the vial of TransFast or Tfx Reagent to room temperature. Dissolve the contents of the vial in 400l of Nuclease-Free Water at room temperature (1mM nal concentration of the cationic lipid component). After adding the Nuclease-Free Water, vortex the sample vigorously for 10 seconds to dissolve the lipid lm. (For Tfx Reagents, place the vial in a 65C water bath for one minute after vortexing. Make sure the level of the water is above the level of the liquid in the vial. Vortex again.) Store the suspended reagent at 20C overnight. Before each use, thaw and vortex the solution. Store any remaining suspended reagent at 20C, where it is stable for 8 weeks. Note: It is necessary to freeze the reagent prior to use. Note: It is normal for the lipid suspension to appear cloudy and contain particulate matter. A slight, residual ring of material may remain in the vial after suspension.
3:1 Charge Ratio of Tfx Reagent:DNA

g DNA/well 0.25 0.50 0.75 1.0
Figure 3.7. Typical optimization matrices for TransFast Reagent and Tfx Reagent:DNA Ratios.
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Chapter 3
Table 3.3. Optimization Protocol Using a 1:1 Charge Ratio of TransFast Reagent to DNA. Amount of DNA Per Well 0.25g Medium (to nal volume) DNA TransFast Reagent* 1,400l 1.8g 5.3l 0.5g 1,400l 3.5g 10.5l 0.75g 1,400l 5.3g 15.8l 1g 1,400l 7.0g 21l
6. Check the transfection efciency using an assay appropriate for the reporter system.
Transfection Protocol for Adherent Cells

After the transfection parameters have been optimized, use the empirically determined conditions for experimental transfections. If you choose not to optimize the transfection parameters, use the general conditions recommended below. Volumes and amounts are given for transfections performed in 60mm plates (values for 100mm plates are given in parentheses). 1. The total volume of medium, DNA and liposome reagent per 60mm dish is 2ml (6ml). To a sterile tube add the appropriate amount of medium, prewarmed to 37C. Add 2.5-10g of plasmid DNA to the medium (7.5-30g) and vortex. We recommend 5g of DNA per 60mm dish (15g), a 1:1 reagent:DNA ratio for TransFast Reagent and a 3:1 reagent:DNA ratio for Tfx Reagents for initial transfection experiments. Add the amount of liposome reagent indicated in Table 3.5 and vortex immediately. Note: The TransFast Reagent and the Tfx Reagents are at a nal concentration of 1mM cationic lipid per suspension. However, the cationic lipid in the Tfx Reagents has two positive charges per molecule while the TransFast Reagent has one positive charge per molecule. Therefore, twice the volume of the TransFast Reagent is required to provide the same charge ratio to DNA.
Table 3.5. Relationship Between Volume of TransFast Reagent and TransFast Reagent:DNA Charge Ratio. Charge Ratio of Liposome Reagent to DNA 1:1 2:1 3:1 4:1 Volume of TransFast Reagent Per g of DNA* 3.0l 6.0l 9.0l 12.0l Volume of Tfx Reagent Per g of DNA 1.5l 3.0l 4.5l 6.0l
* Volumes given are for use with TransFast Reagent suspended in 400l/vial and for use with 24 well plates at 200l/well.
To test 2:1 ratios of TransFast Reagent:DNA, simply double the amount of reagent used for each DNA amount.
Table 3.4. Optimization Protocol Using a 3:1 Charge Ratio of Tfx Reagent to DNA. Amount of DNA Per Well 0.25g Medium (to nal volume) DNA Tfx Reagent* 1,400l 1.8g 7.9l 0.5g 1,400l 3.5g 15.8l 0.75g 1,400l 5.3g 23.6l 1g 1,400l 7.0g 31.5l
* Volumes given are for use with Tfx Reagent suspended in 400l/vial and for use with 24 well plates at 200l/well.
1. For a 24 well plate, the total volume of the medium, DNA and liposome reagent should be 200l per well. The volumes in Tables 3.3 and 3.4 were calculated for seven wells, adequate for 6 replicates for each DNA concentration. In a sterile tube, combine the indicated amount of serum-free medium (prewarmed to 37C) and plasmid DNA and vortex. Add the indicated amount of liposome reagent and vortex immediately. 2. Incubate for 10-15 minutes at room temperature. Incubations longer than 30 minutes result in a lowered transfection efciency. 3. Carefully remove the medium from the cells by aspiration. 4. Briey vortex the liposome reagent/DNA mixture. Add the mixture to the cells (200l per well) and return the plates to the incubator for 1 hour. During the incubation, warm complete medium (cell culture medium containing serum) to 37C. 5. At the end of the 1 hour incubation period, gently overlay the cells with 1ml of complete medium (prewarmed to 37C). Do not remove the transfection medium containing the liposome reagent/DNA mixture. Return the cells to the incubator and continue the incubation for the appropriate length of time before analysis. For many reporter systems (luciferase, CAT and -galactosidase) a 48 hour incubation is sufcient.
*Volumes given are for use with reagents suspended in 400l/vial.
2. Incubate the liposome reagent/DNA mixture for 10-15 minutes at room temperature. 3. Remove the medium from the cells. 4. Add 2ml (or 6ml) of the liposome reagent/DNA mixture to each plate and return the cells to the incubator. Incubate the plates for 1 hour. During the incubation, warm an appropriate volume of serum-containing medium to 37C.
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5. At the end of the incubation period, gently overlay the cells with 4ml (12ml) of the prewarmed medium. Again, do not remove the transfection medium containing the liposome reagent. Return the cells to the incubator and continue incubation for the appropriate length of time before analysis. 6. Check the transfection efciency using an appropriate reporter assay (Figure 3.8). For transient transfection, cells are typically harvested 48 hours after transfection.
experiments; 1 x 106 cells per transfection is usually sufcient. Spin the cells for 5 minutes at 300 x g in a swinging bucket rotor. Resuspend the cell pellet such that the cells are at a concentration of 2 x 106 cells/ml in serum-free media. Re-count the cells and adjust the volume if necessary. 3. Prepare the liposome reagent/DNA mixture. To a sterile tube add the indicated amount of medium (prewarmed to 37C) and DNA to a total volume of 0.5ml and vortex. Add the indicated amount of liposome reagent and vortex immediately (see Table 3.6). 4. Allow the liposome reagent and DNA mixture(s) to incubate for 10-15 minutes at room temperature.
Table 3.6. Optimization Protocols for Suspension Cells. TransFast Reagents (1:1 Charge Ratio) Amount of DNA Per Tube 1g Medium (to nal volume) DNA TransFast Reagent* 0.5ml 1g 3l 2g 0.5ml 2g 6l 3g 0.5ml 3g 9l 4g 0.5ml 4g 12l
Figure 3.8. Histochemical staining of NIH/3T3 cells for -galactosidase activity. NIH/3T3 cells were plated in 24 well plates and transfected with 1g DNA containing the -galactosidase gene under the control of the CMV promoter per well. TransFast Transfection Reagent was used at a 1:1 TransFast Reagent:DNA charge ratio. Cells were xed with glutaraldehyde 2 days post-transfection and stained for -galactosidase activity using standard techniques (See Promega Technical Bulletin #TB097). The cells expressing -galactosidase are stained blue.
* Volumes given are for use with TransFast Reagent suspended in 400l/vial.
Tfx Reagents (3:1 Charge Ratio) Amount of DNA Per Tube 1g Medium (to nal volume) DNA Tfx Reagent* 0.5ml 1g 4.5l 2g 0.5ml 2g 9l 3g 0.5ml 3g 13.5l 4g 0.5ml 4g 18l
Transfection Protocol for Suspension Cells

Optimization of transfection parameters can be performed with suspension cells using the following general guidelines: For 1x106 cells, test 1, 2, 3 and 4g DNA at an initial charge ratio of liposome reagents to DNA of 1:1 for TransFast Reagent and 3:1 for the Tfx Reagents. Incubate for 1 hour in the absence of serum. Additional optimization studies, to test the effect of serum and other liposome reagent:DNA charge ratios can be performed once the optimal amount of DNA has been determined. We recommend also testing a 2:1 charge ratio for both TransFast Reagent and Tfx Reagents for optimization. 1. Suspend the liposome reagent the day before the transfection and store at 20C. 2. On the day of the transfection, determine the cell density using a hemacytometer and spin down enough cells to complete the transfection
*Volumes given are for use with Tfx Reagent suspended in 400l/vial.
5. While the liposome reagent/DNA mixtures are incubating, aliquot 0.5ml of cells (1 x 106 cells) to each well of a 6 well plate. 7. Briey vortex the liposome reagent/DNA mixture and add to the cells (0.5ml/well). Return the cells to the incubator for 1 hour. During the incubation, warm complete medium (containing serum) to 37C. 8. At the end of the incubation period, add 5ml of the prewarmed medium per well. Return the cells to the incubator and continue the incubation for the appropriate length of time before analysis. For many reporter systems (e.g., luciferase, CAT and -galactosidase), a 48 hour incubation is sufcient. 9. Check the transfection efciency using an assay appropriate for the reporter system.
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Chapter 3
Stable Transfections
For stable transfections, cells should be transfected with a plasmid containing a gene for drug resistance, such as neomycin phosphotransferase (see Chapter 6 for details of vectors available from Promega). As a negative control, transfect the cells using DNA that does not contain the drug resistance marker. 1. Prior to transfection, determine the killing concentration of the selective drug being used. 2. Forty-eight hours post transfection, trypsinize adherent cells and re-plate at several different dilutions (for example, 1:100, 1:500) in media containing the appropriate selection drug. 3. For the next 14 days, replace the drugcontaining media every 3 to 4 days. 4. During the second week monitor the cells for distinct islands of surviving cells. Cell death should occur in cultures transfected with the negative control plasmid. 5. Transfer individual clones by standard techniques (e.g., using cloning cylinders) to 96 well plates and continue to maintain cultures in medium containing the appropriate drug. Neomycin (G418) Selection G418 blocks protein synthesis in mammalian cells by interfering with ribosomal function. It is an aminoglycoside, similar in structure to neomycin, gentamycin, and kanamycin (8). Varying concentrations of G418 should be tested as cells differ in their susceptibility to G418. Use 100 to 800 g/ml of G418 in complete medium. G418 should be prepared in a highly buffered solution (eg. 100 mM HEPES, pH 7.3) so that the addition of drug does not alter the pH of the medium. Different lots of G418 can have different potencies, causing many investigators to buy a large amount of one lot to standardize selection conditions. G418 concentration should be calculated using the amount of active drug (usually indicated on each lot label) so that variance is controlled. Cells will divide once or twice in the presence of lethal doses of G418, so the effects of the drug take several days to become apparent. Complete selection can take up to 3 weeks of growth in selective media.
Calculating Stable Transfection Efciency

The following procedure may be used to determine the percentage of stable transfectants obtained. Note: The stained cells will not be viable after this procedure. 1. After approximately 14 days of selection in the appropriate drug, monitor the cultures microscopically for the presence of viable cell clones. When distinct islands of surviving cells are visible and non-transfected cells have died out, proceed with step 2. 2. Prepare stain containing 2% methylene blue in 50-70% methanol 3. Remove the growth media from the cells by aspiration. 4. Add stain to the cells, sufcient to cover the bottom of the dish. 5. Incubate for 5 minutes. 6. Remove the stain and rinse gently under deionized cold water. Shake off excess moisture. 7. Allow the plates to air dry. The plates can be stored at room temperature. 8. Count the number of colonies and calculate the percent of transfectants based on the cell dilution and original cell number. For further information on stable transfections see reference 29.
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Chapter 3
Transfection Protocols Transfectam Reagent for the Transfection of Eukaryotic Cells

Table 3.7. List of Cell Lines Transfected Using Transfectam Reagent. Cell Line Cell Type Fibroblast Embryonic kidney Epthelial Hepatocyte Fibroblast Myoblast Teratocarcinoma Fibroblast Fibroblast Hypophysis Lymphocyte Fibroblast Erythroleukemia Pheochromocytoma Lung epithelial Lung carcinoma Colon adenocarinoma Lymphoblast Leukemic Tlymphoblast Cerebellum neurons Striatium neurons Cortical neurons Astrocytes Adipocytes Anterior pituitary Heart Melantrope cells Chromafn cells
Preparation of Transfectam Reagent Stock Solution

1. Resuspend Transfectam Reagent in 100% ethanol (dehydrated) with vortexing (nal concentration is 2mM) and incubate at room temperature for at least 5 minutes. Overnight storage at 4C ensures complete solubilization and may give improved transfection efciencies. Store the resuspended Transfectam Reagent at 4C, where it is stable for 6 months. Note: For some cells, it may be desirable to minimize the ethanol concentration applied. If so, Transfectam Reagent may be dissolved with vortexing in as little as 1/10 volume of ethanol (dehydrated), incubated at room temperature for 5 minutes, and then further diluted to working concentration in water. 2. Mix the solution before each use. Store the remaining stock at 4C.
Origin
References 43,52-55 56-58 43 59 53,60-63 64 43,65 43,65,66 46 43,67 43 68 69 70 71 72 68 73 68 67 67 67 67 67 43 74 75 43,67 43,67
Established Cell Lines Hamster CHO Human 293 Human HeLa Human Hep G2 Monkey COS Mouse C2C12 Mouse F9 Mouse LM (tk-) Mouse NIH/3T3 Mouse AtT20 Mouse S49 Mouse Balb/3T3 Mouse MEL Rat PC12 Mink Mv1LU Human A549 Human LoVo/Dx Human Human CCRFCEM/VLB Primary Cells Rat Rat Rat Rat Rat Rat Chicken embryo (in vivo) Chicken Pig Bovine
Transfection Protocol for Media Without Serum

We recommend using medium with no added serum for transfection. Some components in serum may degrade the Transfectam Reagent. The presence of albumin, heparin, trypsin or EDTA in the medium also will decrease the efciency of transfection. However, if cell viability is low in medium without serum, use the alternative protocol, provided below. Materials to Be Supplied by the User cell culture medium appropriate for the cell type used The reagent volumes in this protocol are based on use of 60mm culture dishes. Optimization experiments can be performed using adherent cells in 24 well plates. Scale the reagent volumes up or down proportionally if using different sized plates (see Table 3.8).
Table 3.8. Area of Culture Plates for Cell Growth. Growth Area (cm2)a 0.32 1.88 3.83 9.4 8.0 21 55
For further references using Transfectam Reagent in a variety of cell lines, see Appendix A.
Plating Cells
Plate cells the day before the transfection experiment according to the guidelines given in the protocol for Tfx and TransFast Reagents. Suspension cells can be transfected by the following protocol using the equivalent of 106 suspended cells per assay. The volume of reagents can be scaled up or down proportionately depending on the number of cells used per assay.
Size of Plate 96 well 24 well 12 well 6 well 35mm 60mm 100mm

aThis
Relative Areab 0.02 X 0.09 X 0.18 X 0.45 X 0.38 X 1.00 X 2.62 X
information is for Corning culture dishes.
bRelative
area is expressed as a factor of the growth area of the 60mm dish. To determine the approximate plating density, multiply 5 x 105 cells by this factor. To determine the reagent volumes needed for plates other than 60mm plates, multiply the volumes by the appropriate Relative Area factor.
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Chapter 3
1. Add 1-5g of plasmid DNA to 500l of serumfree medium in a sterile tube and vortex (Solution A). We recommend 5g per 60mm dish for the initial tests. 2. For each microgram of plasmid DNA used in Solution A, add between 1.5 and 5l of Transfectam Reagent to 500l of serum-free medium in a sterile tube and mix (Solution B). For the initial tests, use 10l of Transfectam Reagent per 60mm dish per 5g plasmid DNA. 3. Immediately mix Solutions A and B and add directly to the cells. The nal volume will be 1.0ml for a 60mm plate or per 106 suspended cells. 4. Leave in contact with the cells 30 minutes to overnight. Use 2 hours for the initial tests. 5. At the end of the incubation period, gently overlay the cells with 4ml of complete medium with serum (37C). It is not necessary to remove the transfection medium containing the Transfectam Reagent/DNA mixture. Return the cells to the incubator and continue the incubation for the appropriate length of time before analysis. For many reporter assays 48 hours after addition of DNA is sufcient. 6. Check the transfection efciency using the appropriate reporter assay.
4. Leave in contact with the cells 30 minutes to overnight. Use 2 hours for the initial tests. 5. At the end of the incubation period, gently overlay the cells with 4ml of the complete medium (37C). It is not necessary to remove the transfection medium containing the Transfectam Reagent/DNA mixture. Return the cells to the incubator and continue the incubation for the appropriate length of time before analysis. For many reporter assays 48 hours after addition of the DNA is sufcient. 6. Check the transfection efciency using the appropriate reporter assay.
Optimization of Transfection Efciency

Follow these recommendations to obtain the best results possible: Optimize the volume/weight ratio of Transfectam Reagent/DNA in the range of 1.5-5l/g DNA. Ten microliters of Transfectam Reagent stock to 5g DNA is a good initial test for a 60mm plate. Optimize the amount of DNA used in the range of 1-10g DNA. It may not be necessary to increase the quantity of DNA signicantly to obtain optimal results. In fact, if the rst transfection results are satisfactory, a reduced DNA quantity can be tested (while keeping the optimal Transfectam Reagent/DNA ratio constant). The transfection time depends on the specic DNA and cell system used and should be optimized between 30 minutes and overnight. It may be necessary to monitor cell viability if using serum-free medium for a prolonged period because some cells do not thrive under this condition. Usually, the transfection time using the Transfectam Reagent is signicantly shorter than that with standard techniques, reducing the risk of cell death signicantly during transfection. Calibrate the system using a test plasmid with reporter gene function (see Chapter 6).
Transfection Protocol for Medium With Serum

Materials to Be Supplied By the User 0.15M NaCl (sterile) complete medium with serum The reagent volumes in this protocol are based on use of 60mm culture dishes. Scale the reagent volumes up or down proportionately if using different size plates (see Table 3.8). 1. Add 1-5g of plasmid DNA to 50l of 150mM NaCl solution in a sterile tube and vortex (Solution A). We recommend 5g per 60mm dish for the initial tests. 2. For each microgram of plasmid DNA used in Solution A, add between 1.5 and 5l of Transfectam Reagent to 50l of 150mM NaCl solution in a sterile tube and mix (Solution B). For the initial tests, use 10l of Transfectam Reagent per 60mm dish, for 5g plasmid DNA. 3. Immediately mix solutions A and B, wait 10 minutes, then add to the cells.
Tip Studies by Boukhnikachvili et al. suggest that the efciency of transfection using DOGS (Transfectam Reagent) is related to the structure of the lipid/DNA complex formed, and increasing both pH and ionic strength (76) can increase formation of such complexes.
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Notes
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Chapter 3
Notes
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Chapter 4
Introduction
The ProFection Mammalian Transfection Systems offer the choice of calcium phosphate or DEAEDextran mediated transfection procedures. Both of these methods appear to facilitate DNA binding to cell membranes and entry of the DNA into the cell via endocytosis. Calcium phosphate also appears to provide protection against intracellular and serum nucleases (77). Calcium phosphate transfection may be used for the production of long-term stable transfectants, works well for transient expression of transfected genes and can be used with most adherent cell lines. DEAE-Dextran transfection is also an efcient method for introducing DNA into many cell types, including some cell suspensions. However, its suitability is limited to transient expression studies and it is not recommended for the production of stable transfectants (10). For transient expression studies using a particular cell type, both protocols should be tried in order to determine the most efcient method. For a list of references using the ProFection Mammalian Transfection Systems in a variety of cell lines, see Appendix A. Factors That Affect Efciency of Gene Transfer Transfection efciencies can be increased in many cell types by additional treatments after the primary exposure of the cells to calcium phosphate-DNA or DEAE-Dextran and DNA. The most effective and routinely used agents are glycerol (78,79), dimethyl sulfoxide (DMSO) (79-81), chloroquine (82) and sodium butyrate (83). Since each of these chemicals is toxic to cells, the conditions for transfection of individual cell types must be carefully optimized for reagent concentration and exposure time.
Figure 4.1. CHO cells were transfected with the ProFection Mammalian Transfection System-Calcium Phosphate and DNA containing the green uorescent protein reporter gene. Cells were counter-stained with propidium iodide.
Figure 4.2. NIH /3T3 cells were transfected with the ProFection Mammalian Transfection System-Calcium Phosphate and DNA containing the green uorescent protein reporter gene.
Transfection Protocol
The protocol given below is for a 60mm plate. For different size plates scale the volumes and amounts proportionally according to the information given in Chapter 3, Table 3.8. 1. Plate the cells the day before transfection as described in Chapter 3. 2. Three hours prior to transfection, remove the medium from the cells and replace it with fresh growth medium. 3. Thaw all system components and warm them to room temperature. Mix each component thoroughly by swirling the container or vortexing.
Calcium Phosphate-Mediated Transfection

A precipitate containing calcium phosphate and DNA is formed by slowly mixing a HEPES-buffered phosphate solution with a solution containing calcium chloride and DNA. These DNA precipitates are then taken into eukaryotic cells by an endocytictype mechanism.
Plating Cells for Transfection

Plate cells the day before the transfection experiment according to the guidelines given in Chapter 3.
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Chapter 4
4. For each transfection, prepare the DNA and 2X HBS solutions in separate sterile tubes. Add the DNA and water to the rst tube, mix well, then add the CaCl2 and mix again. Add the specied amount of 2X HBS to the second tube. per 60mm dish 6-12g 37l to nal volume 0.3ml 0.3ml
Glycerol or DMSO Shock

Glycerol Shock
High transfection efciencies can be obtained by leaving the DNA/calcium phosphate solution on the cells until the cells are harvested or selective pressure is applied. HeLa cells, for example, respond well to this treatment. However, transfection of some cell lines, such as CHO cells, is enhanced by a glycerol shock step. The glycerol shock step may be performed 4-16 hours after transfection. In general, if cells can tolerate the calcium phosphate solution, it is best to leave it on for as long as possible and perform the glycerol shock 16 hours after transfection. Cell lines that are more sensitive to the calcium phosphate solution may respond better to a glycerol shock step performed earlier, such as 4 hours after exposure to the DNA. Do not expose the cells to the glycerol solution for more than 2 minutes. The optimum time interval before performing the glycerol shock should be determined empirically for each cell line. Materials to Be Supplied by the User (Solution compositions are provided at the end of this chapter.) glycerol shock solution (15% glycerol) wash solution: 1X PBS or 1X HBSS 1. Prepare a fresh glycerol shock solution in 1X HBS and warm it to 37C, along with growth medium and wash solution. 2. Wash the cells once with 5ml of wash solution per 60mm plate. 3. Add 2ml of the glycerol shock solution per 60mm plate. 4. Incubate for up to 2 minutes at room temperature. 5. Remove the glycerol shock solution and wash the cells twice with 5ml of wash solution per 60mm plate. 6. Add regular growth medium and return the cells to a 37C incubator.
DNA 2M CaCl2 sterile, deionized water 2X HBS
5. Working in a tissue culture hood, gently vortex the tube containing the 2X HBS solution. The speed should be adjusted such that the tube can be vortexed safely with the cap off and can accommodate the addition of the prepared DNA solution. Continue to vortex while slowly adding the prepared DNA solution dropwise to the 2X HBS. (Alternatively, bubble air with a pipet through the 2X HBS while slowly adding the CaCl2/DNA solution). When the DNA addition is complete, the solution should appear slightly opaque due to the formation of a ne calcium phosphate-DNA coprecipitate. Incubate the solution at room temperature for 30 minutes. 6. Vortex the transfection solution again just prior to adding it to the cells. Add the solution dropwise to the plates. Swirl the plates to distribute the precipitate evenly over the cells. Return the plates to a 37C CO2 incubator. 7. When working with sensitive cells, the culture medium should be changed 4-16 hours after transfection. The length of the incubation should be optimized for individual cell lines. Primary cells are particularly sensitive and should not be exposed to calcium phosphate for more than 4 hours. 8. In general cells may be harvested or selective media applied 48-72 hours after transfection.
Tip Strontium chloride can be used in place of calcium chloride if the cells being transfected are sensitive to the high calcium concentration present in the calcium phosphate/DNA precipitate (84). Tip To increase efciency of transfection of some cell types, glycerol may be added during incubation with the calcium phosphate/ DNA precipitate (85).
DMSO Shock
Certain cell types exhibit enhanced transfection efciencies after exposure to dimethyl sulfoxide (DMSO). The DMSO step can be added to either the calcium phosphate or DEAE-Dextran transfection protocols. DMSO, like glycerol, is toxic to cells and the concentration and exposure times require careful optimization for each cell type. Most cells should not be exposed to DMSO for more than 2.5 minutes. One representative protocol for a DMSO shock is provided.
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Chapter 4
Materials to Be Supplied by the User (Solution compositions are provided at the end of this chapter.) DMSO shock solution (10% DMSO) 1. Remove the medium from the cells. 2. Immediately before use, prepare the DMSO shock solution and warm it to 37C. Prepare 2ml per 60mm plate. 3. Add the DMSO shock solution to the cells and incubate for 2.5 minutes. Do not return the plates to an incubator during this time. 4. Remove the DMSO shock solution, wash the cells twice and add 5ml of regular growth medium per 60mm plate. Return the cells to the 37C incubator.
Materials to Be Supplied by the User (Solution Compositions are provided at the end of this chapter.) wash solution: 1X PBS or 1X HBSS optional: 8mM chloroquine (in sterile water)
Standard DEAE-Dextran Protocol

The protocol given below is for a 60mm plate. For different size plates scale the volumes and amounts proportionally according to the information given in Chapter 3, Table 3.8. 1. Plate cells the day before the transfection experiment according to the guidelines given in Chapter 3. 2. Prepare the wash solution (1X PBS or 1X HBSS) and warm it to 37C. Ten milliliters of wash solution are required for each 60mm plate. Warm the DEAE-Dextran solution to 37C. 3. Dilute the 10X PBS stock 10-fold with sterile water. You will need approximately 0.4ml of 1X PBS per 60mm plate. Prepare the transfection solutions as outlined below: Per 60mm dish: Using a sterile tube, dilute 2-6g DNA to a nal volume of 326l in 1X PBS. Add 17l of the 10mg/ml DEAE-Dextran and mix by gently tapping the tube. 4. Remove the medium from the cells. Wash the cells twice with wash solution using 2 x 5ml per 60mm plate. 5. Add the DNA/DEAE-Dextran mixture and disperse it evenly over the cells. The nal concentration of DEAE-Dextran in the salt solution is approximately 0.5mg/ml. 6. Incubate the plates at 37C for 30 minutes. Rock the plates occasionally to keep the cells moist. 7. Gently add 3.5ml of growth medium per 60mm plate. Incubate up to 2.5 hours at 37C or until cytotoxicity is apparent. Gently change the medium or follow with a DMSO shock. Optional: Add 35l of 8mM chloroquine per 60mm plate along with the medium during the 2.5 hour incubation step. If chloroquine is added, the culture medium must be replaced after 4 hours (or earlier, if signs of cytotoxicity are apparent). The time that the cells are exposed to chloroquine must be empirically determined for each cell line. 8. Harvest the cells 48 hours after transfection.
DEAE-Dextran-Mediated Transfection
DEAE-Dextran, a polymeric cation, associates tightly with the negatively charged DNA and carries it into the cell. As DEAE-Dextran is toxic to cells, transfection conditions for individual cell lines may require careful optimization of both DEAE-Dextran concentration and exposure times. At higher DEAEDextran concentrations, the exposure time to cells can be shortened in order to minimize cell death. This protocol may be inappropriate for certain cell lines for which DEAE-Dextran is highly toxic. Two different protocols for DEAE-Dextran transfections are given. The standard protocol involves concurrent exposure of cells to DEAEDextran and DNA. The second protocol involves pretreatment of the cells with DEAE-Dextran and is a modication of the procedure described by Al-Molish, et al. (86). It offers the advantages of limited DEAE-Dextran exposure and longer DNA incubation, allowing maximal DNA uptake. The best protocol for a particular cell line should be determined experimentally. The addition of 80M chloroquine along with the DNA is an option for both protocols. For some cell lines, chloroquine dramatically increases transfection efciencies; for others, it has a minimal effect and may be quite cytotoxic. The optimal amount of DNA to use for transfection will vary with the cell line and type of reporter construct being used. Generally, 2-6g of DNA will be sufcient for a 60mm plate and 4-10g DNA will be sufcient for a 100mm plate. Both protocols require a sterile calcium- and magnesium-free salt solution for the wash steps. This wash solution is not provided with the system. 1X PBS or another salt solution such as 1X HBSS works well for this purpose.
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Chapter 4
DEAE-Dextran Pretreatment Protocol

1. Plate the cells the day before the transfection experiment as described in Chapter 3. 2. Prepare the wash solution and warm it to 37C. Fifteen milliliters of wash solution are required for each 60mm plate. 3. Dilute the 10X PBS stock 10-fold with sterile water. You will need approximately 3ml of 1X PBS per 60mm plate. Prepare the transfection solutions as outlined below: Dilute the DEAE-Dextran stock solution 1:10 in the 1X PBS solution prepared above. You will need 2ml of diluted DEAE-Dextran per 60mm plate. Dilute the DNA in 1X PBS to a nal volume of 325l for a 60mm plate. 4. Remove the medium from the cells. Add 5ml of sterile wash solution for 60mm plates. Incubate for 15 minutes at room temperature. 5. Remove the wash solution from the cells. Add 2ml of the diluted DEAE-Dextran solution per 60mm plate. Incubate for 9 minutes at room temperature. 6. Remove the DEAE-Dextran solution. Very gently wash the cells twice with 2 x 5ml of wash solution per 60mm plate. Be careful not to dislodge the cells, which may begin to detach after exposure to DEAE-Dextran. 7. Remove the nal wash. Add the diluted DNA and disperse it evenly over the cells. Incubate for 30 minutes in a 37C CO2 incubator. Rock the plates occasionally to keep the cells moist. 8. Add 3.5l of regular growth medium per 60mm plate. Optional: Add 35ml of 8mM chloroquine per 60mm plate together with the medium. If chloroquine is added, the culture medium must be replaced after 4 hours (or earlier, if signs of cytotoxicity are apparent). The time that the cells are exposed to chloroquine must be empirically determined for each cell line. 9. Return the plates to a 37C CO2 incubator. 10. Harvest the cells 48 hours after transfection.
Composition of Buffers and Solutions

2X HBS (HEPES-Buffered Saline) 50mM HEPES (pH 7.1) 280mM NaCl 1.5mM Na2HPO4 The nal pH should be 7.1 1X PBS (Phosphate Buffered Saline) 137mM NaCl 2.7mM KCl 4.3mM Na2HPO4 1.47mM KH2PO4 The nal pH should be 7.1 1X HBSS (Hanks Balanced Salt Solution) 5mM KCl 0.3mM KH2PO4 138mM NaCl 4mM NaHCO3 0.3mM Na2HPO4 5.6mM D-glucose The nal pH should be 7.1. 1X Trypsin-EDTA solution 0.05% (w/v) trypsin 0.53mM EDTA Dissolve these components in a calcium- and magnesium-free salt solution such as 1X PBS or 1X HBSS. TE buffer 10mM 1mM
Tris-HCl (pH 8.0) EDTA
Glycerol shock solution 1X HBS 15% Glycerol DMSO shock solution 1X PBS 10% DMSO (tissue culture grade)
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Notes
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Chapter 5
General Troubleshooting Tips

Symptoms No transfection or low transfection efciency Variable transfection in replicate cultures experiments Possible Causes Poor quality DNA Comments The DNA should be puried on CsCl gradients or equivalent methods. The A260:A280 ratio of the DNA should be 1.8 or greater. Test cultures for Mycoplasma contamination. Destroy contaminated cultures and start a new culture from a fresh stock. Transfection efciency may decrease if cells have been passaged for many generations. Start a fresh culture from cell stocks that were frozen at an early passage. Some cells, particularly lymphocytes, will exhibit variability in transfection efciency if they are left in culture beyond 1-2 weeks. Maintain a consistent cell density at the time of transfection for each experiment.
Cells are contaminated with Mycoplasma Suboptimal growth of cells
Variable cell density
Cationic Lipid Reagent Troubleshooting

Symptoms No transfection or low transfection efciency Possible Causes Charge ratio of reagent to DNA is sub-optimal Excessive cell death Comments Optimize the reagent to DNA charge ratio. Charge ratios of 1:1, 2:1, 3:1 and 4:1 work well for many cell lines, but ratios outside this range may be optimal for a particular cell type or application. Decrease the time of exposure of the cells to the reagent. Lower the amount of input DNA and cationic lipid reagent, while holding the charge ratio constant. Increase cell density for the transfection step. Remove cationic lipid reagent/DNA mixture from the cells after the transfection period and prior to adding complete medium.
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Chapter 5
Calcium Phosphate Transfection Troubleshooting

Symptoms No transfection or low transfection efciency Possible Causes Poor precipitate formation Comments The CaCl2/DNA and 2X HBS solutions should be at room temperature (22-25C) when they are mixed. Higher or lower temperatures for precipitate formation can lead to decreased transfection efciency. The addition of CaCl2/DNA to the 2X HBS solution should be performed dropwise and with continuous mixing. The concentration of DNA can affect the size of the precipitate. Low amounts of DNA (less than 1g) can be supplemented with sheared carrier DNA such as salmon or herring sperm DNA. However, there are conicting reports in the literature as to the efcacy of adding carrier DNA (80,87). The precipitate should be added dropwise around the dish to the medium bathing the cells, and the medium should be mixed thoroughly at the end of the addition. This helps to evenly distribute the precipitate and avoid the localized acidication of cells. After the addition of the calcium phosphate precipitate to the cells, the pH of the medium should be between 7.2 and 7.4. The CO2 concentration in the incubator should be maintained at an appropriate level (generally 5-10%, depending on the composition of the culture medium). The pH of the HBS solution should be 7.1. A large volume of added DNA in Tris buffer could change the pH. The DNA should be resuspended in water, 1mM Tris or, if present in 10mM Tris, should be fairly concentrated so that a relatively small volume of the DNA solution is added to the HBS. pH of the 2X HBS may have changed on storage. Check the pH and adjust it to 7.1 if necessary.
pH not optimal
DEAE-Dextran Transfection Troubleshooting

Symptoms No transfection or low transfection efciency Possible Causes Excessive cell death Comments Decrease the concentration of DEAE-Dextran or shorten the time during which the cells are exposed to DEAE-Dextran. Decrease the time of exposure to chloroquine. Certain types of cells that are very sensitive to DEAEDextran toxicity, such as primary cell cultures, may require a higher cell concentration at the time of transfection. For some cell lines, lower concentrations of DNA can be used for standard DEAE-Dextran transfections compared to calcium phosphate transfection. Establish a dose-response curve to determine the optimal DNA concentration to use.
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Notes
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Chapter 6
Introduction
Genetic reporter systems have contributed greatly to the study of eukaryotic gene expression and regulation. Although reporter genes have played a signicant role in numerous applications (88), they are most frequently used as indicators of transcriptional activity in cells (89). Typically, a reporter gene is joined to a promoter sequence in an expression vector that is transfected. Following transfer, the cells are assayed for the presence of the reporter by directly measuring the amount of reporter mRNA, the reporter protein itself or the enzymatic activity of the reporter protein. An ideal reporter gene is not endogenously expressed in the cell type of interest, and is amenable to assays that are sensitive, quantitative, rapid, easy, reproducible and safe. The most popular systems for monitoring genetic activity in eukaryotic cells include chloramphenicol acetyltransferase (CAT), -galactosidase, rey luciferase, growth hormone (GH), -glucuronidase (GUS), alkaline phosphatase (AP) and, most recently, green uorescent protein (GFP) and Renilla luciferase (90,91) A control vector can be used to normalize for transfection efciency or cell lysate recovery between treatments or transfection experiments (92). Typically, the control reporter gene is driven by a strong, constitutive promoter and is cotransfected with experimental vectors. The experimental regulatory sequences are linked to a different reporter gene so that the relative activities of the two reporter gene products can be assayed individually. Control vectors can also be used to optimize transfection methods. Gene transfer efciency is typically monitored by assaying reporter activity in cell lysates, or by staining the cells in situ to estimate the percentage of cells expressing the transferred gene (80).
luciferase reporter especially suited for transient assays designed to assess inducible and short-lived effects. The rey luciferase enzyme catalyzes a reaction using D-luciferin and ATP in the presence of oxygen and Mg2+, resulting in light emission. The total amount of light measured during a given time interval is proportional to the amount of luciferase reporter activity in the sample. The assay has been improved by including coenzyme A in the reaction, which provides a longer, sustained light reaction with greater sensitivity (97). Light emission is typically quantied over a dened assay period. The extended glow reaction of the enhanced luciferase assay allows for accurate measurement of the luminescence reaction when using a luminometer or scintillation counter. The sensitivity of the luciferase assay is in the subattomole range, approximately 30-1,000 times greater compared to the sensitivity of CAT assays (96). An added advantage is that luciferase assay results can be obtained in minutes compared to hours, or even days, for the radioactive CAT assay. The linear range of the rey luciferase assay extends over an impressive 8 orders of magnitude of rey luciferase concentration. Dual-Luciferase Reporter Assay System Promegas Dual-Luciferase Reporter (DLR) Assay System combines the speed, sensitivity and convenience of two luciferase reporter enzymes into an integrated, single-tube, dual-reporter assay format. The DLR Assay is designed to provide rapid, sequential quantitation of rey luciferase and sea pansy (Renilla reniformis) luciferase in cell lysates or cell-free translation systems. Because the rey and Renilla luciferases are of distinct evolutionary origins, they have dissimilar enzyme structures and substrate requirements. These differences make it possible to selectively discriminate between their respective bioluminescent reactions. Thus, the luminescence from the rey luciferase reaction may be quenched while simultaneously activating the luminescent reaction of Renilla luciferase (the control reporter).
Promegas Reporter Systems

Promega currently offers reporter vectors and assay systems for chloramphenicol acetyltransferase (CAT), -galactosidase, rey luciferase, and an integrated dual-reporter assay system for the sequential quantitation of rey and Renilla (sea pansy) luciferases. Luciferase Reporter Assay System The luciferase enzyme used most frequently for reporter gene technology is derived from the coding sequence of the luc gene cloned from the rey Photinus pyralis (91,93,94). Compared to CAT, the rey luciferase protein has a shorter half-life in transfected mammalian cells (95,96), making the
Renilla luciferase is a 36kDa monomeric protein that utilizes oxygen and coelenterate luciferin (coelenterazine) to generate light emission (98). In the integrated Dual-Luciferase Reporter Assay chemistry, the kinetics of the Renilla luciferase reaction provide a glow-type luminescent signal that decays slowly over the course of the measurement. Similar to rey luciferase, the luminescent reaction catalyzed by Renilla luciferase provides high sensitivity and a linear range extending over 7 orders of magnitude of Renilla luciferase concentration.
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39
Chapter 6
CAT Reporter Assay System The CAT gene is derived from transposon 9 of E. coli (99). CAT is a trimeric protein comprising three identical subunits of 25kDa (100). The CAT protein is relatively stable in mammalian cells, although the mRNA has a comparatively short half-life, making the CAT reporter especially suited for transient assays designed to assess accumulation of expressed protein (95). CAT catalyzes the transfer of the acetyl group from acetyl-CoA to the substrate, chloramphenicol. The enzyme reaction can be quantitated by incubating cell lysates with [14C]chloramphenicol and following product formation by physical separation with thin layer chromatography (TLC) or organic extraction (101,102). -Galactosidase Reporter Assay System The E. coli lacZ gene encodes -galactosidase, a tetrameric enzyme that catalyzes the hydrolysis of -galactoside sugars such as lactose. The enzymatic activity in cell extracts can be assayed with various specialized substrates that allow quantitation of enzyme activity using a spectrophotometer, a uorometer or a luminometer. A major strength of this reporter gene is the ability to easily assay in situ expression with histochemical staining (see Figure 6.1). The -galactosidase reporter gene is frequently used as a control vector for normalizing transfection efciency when co-transfected with chimeric DNAs linked to other reporter genes (92). One potential limitation of this reporter gene is that certain mammalian cells have endogenous lysosomal -galactosidase activity. Enzyme assays performed at a higher pH of 7.3-8.0, or with cell extracts pre-heated to 50C, preferentially favor the E. coli enzyme (88,103). However, because of endogenous
A. HeLa Cells
cellular -galactosidase activity, it is important to include negative control extracts or cells that have not been transfected as comparisons for the cellfree and in situ analyses.
Firey Luciferase Reporter Gene Systems

Firey Luciferase Reporter Vectors
Promegas Luciferase pGL2 and pGL3 Reporter Vectors and Luciferase Assay Reagents provide a basis for rapid, quantitative analysis of factors that potentially regulate gene expression. The pGL2 and pGL3 Luciferase Reporter Vectors contain the cDNA encoding luciferase (luc) cloned from the North American rey (Photinus pyralis), as well as numerous features that aid in the characterization and manipulation of cloned regulatory sequences. Changes in luciferase reporter activity directly correlate to the transcriptional activity of the cloned regulatory element when expressed in transfected cells. All pGL3 Vectors contain a modied rey luciferase cDNA, designated luc+, and a vector backbone that has been designed to provide enhanced reporter gene expression (Figure 6.2). These modications help to ensure that the luciferase reporter gene, itself, does not contribute spurious transcriptional signals. Further details on these modications are provided in Technical Manual #TM033. The combination of modications embodied in the pGL3 Vector family provides greater exibility in performing genetic manipulations, minimal background activity and luciferase expression levels that are dramatically higher than previously obtained with the pGL2 Reporter Vectors (104). Using the pGL3 Vectors, it is now possible to obtain
B. BHK Cells
Figure 6.1. Histochemical staining of HeLa and BHK cells for -galactosidase activity. HeLa (Panel A) and BHK (Panel B) cells were plated in 24 well plates and transfected with pCI- lacZ vector DNA. BHK cells were transfected with Tfx-10 Reagent at a 2:1 Reagent:DNA ratio, with 1,000ng of DNA. HeLa cells were transfected with Tfx-20 Reagent at a 2:1 Reagent:DNA ratio, with 250ng of DNA. The transfections were performed in the absence of serum for one hour. Cells were xed with glutaraldehyde 48 hours post-transfection and stained for -galactosidase using standard techniques. The cells expressing -galactosidase are stained blue. 40 Transfection Guide
Chapter 6
measurable luciferase expression in cell types that are difcult to transfect, when studying weak promoter elements, or when performing in vivo luminescence measurements. It is important to recognize that absolute light unit values and relative expression proles of reporter vectors will vary between different cell types (104). The appropriate control vector should always be included in experiments utilizing genetic reporter systems.
160 Luciferase Assay System 120
Light Intensity
80
40 Conventional Method
General Considerations for Firey Luciferase Reporter Assays

Promegas rey Luciferase Assay System offers several advantages over conventional assays for luciferase (97). The reaction catalyzed by rey luciferase is oxidation of beetle luciferin with concomitant production of a photon. Under conventional reaction conditions, the oxidation occurs from an enzyme intermediate, luciferyl-AMP. However, recent investigation has revealed that coenzyme A is a substrate in the luminescent reaction. In the presence of CoA, oxidation occurs presumably through luciferyl-CoA. The result is light production without the characteristic self-inhibition of luciferase observed in other assays (97). The conventional assay for luciferase generates a ash of light that rapidly decays after enzyme and substrate are mixed, thus requiring automated injection luminometers for measurements of photon production. Promegas Luciferase Assay System
0 0 10 20 30 40 50 60
Time (seconds)
Figure 6.3. Comparison of Promegas Luciferase Assay System to the conventional luciferase assay method.
allows for greater enzymatic turnover of luciferase (97), which results in greater light intensity that is nearly constant for measurements of up to several minutes (Figure 6.3). The constant light intensity generated in Promegas assay eliminates the need for rapid mixing protocols and automated injection devices. The simplied assay procedure is adaptable to different measurement methods for light production, such as scintillation counting or exposure to photographic lm. Please request Promega Technical Bulletins #TB101 or #TB161 for a detailed Luciferase Reporter Assay Protocol.
Ampr
Synthetic poly(A) signal / transcriptional pause site (for background reduction) f1 ori
Ampr 5 11 15 21 28 32 36 53 f1 ori
Synthetic poly(A) signal / transcriptional pause site (for background reduction)
ori
pGL3-Basic Vector
(4818bp)
Kpn I Sac I Mlu I Nhe I Sma I Xho I BgI II Hin d III Nco I 86
ori
pGL3-Promoter Vector
(5010bp)
Kpn I Sac I Mlu I Nhe I Sma I Xho I BgI II

SV40 Promoter
5 11 15 21 28 32 36
2010 Sal I 2004 Bam H I SV40 late poly(A) signal (for luc+ reporter) Hpa I 1902
luc+
2202 Sal I 2196 Bam H I SV40 late poly(A) signal (for luc+ reporter) Hpa I 2094
Nar I 121
Hin d III 245

luc+
Nco I 278
Xba I 1934 Xba I 1742
Ampr
Ampr
ori
pGL3-Enhancer Vector
(5064bp)
Kpn I Sac I Mlu I Nhe I Sma I Xho I BgI II Hin d III
5 11 15 21 28 32 36 53
ori
pGL3-Control Vector
2448 Sal I 2442 Bam H I SV40 Enhancer (5256bp)

SV40 Promoter
5 11 15 21 28 32 36
2256 Sal I 2250 Bam H I
luc+
Nco I 86
Nar I 121
Hin d III 245

luc+
SV40 Enhancer
SV40 late poly(A) signal (for luc+ reporter) Hpa I 1902
SV40 late poly(A) signal (for luc+ reporter) Hpa I 2094
Nco I 278 Nar I 313
Xba I 1934 Xba I 1742
Figure 6.2. Circle maps of the pGL3 Vectors.
Transfection Guide
41
Chapter 6
Dual-Luciferase Reporter Assay System

pRL Renilla Luciferase Vectors
The pRL family of Renilla luciferase vectors, used with the Dual-Luciferase Reporter Assay System, provides options for Renilla luciferase expression in transfected mammalian cells. The pRL Vectors may be used in combination with any experimental rey luciferase vectors to co-transfect mammalian cells. Thus, the expression of Renilla luciferase can provide an internal control value to which expression of the experimental rey luciferase reporter gene may be normalized. The pRL family of control reporter vectors contain the cDNA encoding Renilla luciferase (Rluc) cloned from the anthozoan coelenterate Renilla reniformis, the sea pansy (105), with some minor modications for convenience as a genetic reporter. The constitutive expression of Rluc is provided by one of several available promoter elements. The pRL Vectors are currently available in three promoter congurations and one promoter-less conguration (Figure 6.4). General Considerations for Co-Transfection Experiments The pRL Vector of choice may be used in combination with any experimental reporter vector to co-transfect mammalian cells. However, it is important to realize the potential for trans effects between co-transfection
promoters that may affect reporter gene expression (106). Primarily, this is of concern when working with very strong promoter/enhancer elements resident on one or the other, or both, of the control and experimental reporter vectors. The occurrence and magnitude of such effects will depend on 1) the combination and activities of the genetic regulatory elements present on the co-transfected vectors; 2) the relative ratio of experimental vector to control vector introduced into the cells; and 3) on the cell type itself. To help ensure independent genetic expression between experimental and control reporter genes, perform preliminary co-transfection experiments to optimize both the amount of vector DNA and the ratio of co-reporter vectors added to the transfection mix. The extreme sensitivity of both the rey and Renilla luciferase assays, and the very large linear range of luminometers (typically 5-6 logs) allows accurate measurement of substantially different experimental and control luminescence values. Therefore, relatively small quantities of a pRL coreporter vector are needed to provide low-level, constitutive expression of Renilla luciferase control activity. Ratios of luciferase co-transfection vectors of 50:1 or greater are feasible, and in some instances will be preferable to aid in suppressing trans effects between promoter elements.
Ampr
ori
Ampr 2257 Bam H I SV40 late poly(A)
ori
1883 Bam H I SV40 late poly(A) 1631 Xba I Rluc
pRL-SV40 Vector (3705bp)
Bgl II 1 Kpn I 58
SV40 Early Enhancer/Promoter
2005 Xba I
pRL-CMV Vector (4079bp)

Rluc T7 Promoter
Bgl II 1
T7 Promoter
Hin d III 420 Pst I 462
CMV Immediate Early Enhancer/Promoter
Nhe I 684 Csp45 I 700
1074 Csp45 I
Pst I 836 Nhe I 1058
Eco ICR I 725 Hin d III 754
Ampr 2223 BamH I SV40 late poly(A) 1971 Xba I

ori
Ampr
ori
pRL-TK Vector (4045bp)
Bgl II 1
pRL-null Vector (3320bp)

1498 Bam H I T7 Promoter Rluc
Rluc T7 Promoter
HSV TK Promoter
SV40 late poly(A) 1246 Xba I
Nhe I 299 Csp45 I 315
Bgl II Xho I Sac I Eco ICR I Hind III Nde I Nsi I Sph I Spe I Nar I Sal I Mlu I EcoR I Xma I Sma I Pst I
1 5 12 10 15 24 30 32 34 41 47 51 58 65 67 77
(1040) Csp45 I
EcoR I 649 Hin d III 760 Nhe I 1024
Figure 6.4. Circle maps of the pRL-SV40, pRL-CMV, pRL-TK and pRL-null Vectors.
42
Transfection Guide
Chapter 6
General Considerations for the Dual-Luciferase Reporter Assay System The luminescent signal from each of the two luciferase reporter enzymes may be quantitated immediately following lysate preparation without the need for dividing samples or performing additional treatments. The rey luciferase reporter assay is initiated by adding an aliquot of lysate to Luciferase Assay Reagent II. Quenching of rey luminescence, and concomitant activation of the Renilla luciferase, is accomplished by adding Stop & Glo Reagent to the sample tube immediately upon completion of the rey reaction. Luminescence signal from the rey reaction is quenched by at least a factor of 100,000 (to 0.001% residual light output) within one second following the addition of Stop & Glo Reagent (see Figure 6.5). Complete activation of Renilla luciferase is also achieved within this one-second period. When using a manual luminometer, the time required to quantitate both luciferase reporter activities will be approximately 30 seconds.
Please request Promega Technical Manuals #TM040 or #TM046 for more information on the Dual-Luciferase Reporter Assay System protocol.
CAT Reporter Gene Systems

pCAT Reporter Vectors
Promega provides two families of CAT gene reporter vectors: pCAT and pCAT 3 Reporter Vectors. Each family of vectors contains four plasmids which are referred to as pCAT- or pCAT 3-Basic (lacking eukaryotic promoter and enhancer sequences), Enhancer (with the SV40 early enhancer element 3 of the CAT gene), Promoter (with the SV40 early promoter driving expression of the CAT gene) and Control (with the SV40 early promoter driving expression of the CAT gene and the SV40 early enhancer 3 of the gene). Figure 6.6 provides vector maps for the pCAT 3 Vectors; maps for the pCAT Vectors are provided in Technical Bulletins #TB080083. The increase in CAT expression observed with the pCAT 3 Vectors provides greater sensitivity. It may now be possible to obtain measurable CAT expression in cell types that are difcult to transfect or when studying weak promoter elements. Users of the pCAT and pCAT3 Vectors should be aware, however, that relative expression proles vary between different cell types (107). Therefore, it is important to include the appropriate control vectors in all experiments. Details on pCAT3 Reporter Vector cloning strategies and analyses are provided in Technical Manual #TM036.
1,000,000
100,000
80,600
116,800
10,000
Relative Light Units
1,000
100
10
0.0004% Residual Activity
0.10
0.28 Firefly Luciferase Activity Firefly Luciferase Activity after Quench
Renilla Luciferase Activity
CAT Enzyme Assays

Chloramphenicol acetyltransferase (CAT), encoded by a bacterial drug-resistance gene, inactivates chloramphenicol by acetylating the drug at one or both of its two hydroxyl groups (108). This gene is not found in eukaryotes, and therefore eukaryotic cells contain no background CAT activity. This characteristic, along with the sensitivity of the assay for CAT activity, has made the CAT gene the most widely used reporter gene for studies of mammalian gene expression (90,109). CAT activity may be monitored by two alternative methods using Promegas CAT Enzyme Assay System. Please request Promega Technical Bulletin #TB084 for a detailed CAT assay protocol.
Figure 6.5. Measurement of luciferase activities before and after the addition of Stop & Glo Reagent.
Instrument Considerations
The Turner Designs Model TD-20/20 Luminometer (Promega Cat.# E2061) is ideally suited for lowthroughput processing of Dual-Luciferase Reporter Assays. The instrument is pre-programmed to complete sequential readings of both rey and Renilla luciferase reporter activities with a single Start command. Further, the instrument is programmed to provide the individual and normalized luciferase values, as well as statistical analyses of values measured within replicate groups.
Transfection Guide
43
Chapter 6
-Galactosidase Reporter Gene System

The enzyme -galactosidase is widely used as a reporter molecule for both in vitro and transgenic applications. Promegas pSV--Galactosidase Control Vector (Cat.# E1081; Figure 6.7) is designed as a positive control vector for monitoring transfection efciencies of mammalian cells. The SV40 early promoter and enhancer drive transcription of the bacterial lacZ gene, which encodes -galactosidase. -Galactosidase is an excellent reporter enzyme (89,110) that can be assayed quickly and directly in cell extracts using a spectrophotometric assay (111), or in xed cells by in situ staining (29). A protocol for histochemical staining for -Galactosidase can be found in Promega Technical Bulletin #TB097.
Eco R I 6815
Hin d III 414

SV40 Promoter and Enhancer
lac Z
pSV--Galactosidase
ori
Vector (6821bp)
Amp r
Eco R I 3701 Bam H I 4151 Sal I 4163 Pst I 4173
Figure 6.7. Circle map of the pSV--Galactosidase Control Vector.
Ampr f1 ori
Synthetic poly(A) signal and transcriptional pause site (for background reduction)
Ampr f1 ori
pCAT3-Basic Vector
(4047bp)
Kpn I Sac I Mlu I Nhe I Sma I Xho I BgI II (Hin d III)
5 11 15 21 28 32 36 53 ori
pCAT3-Promoter Vector
(4239bp)
ori
CAT
(Hin d III) 276 Nco I 309

1431 Sal I 1425 Bam H I
SV40 Promoter (Hin d III) 245
5 11 15 21 28 32 36
CAT
1239 Sal I 1233 Bam H I
SV40 Hpa I 1131 late poly(A) region
Xba I 971
Hpa I 1323
SV40 late poly(A) region
Xba I 1163
Ampr f1 ori
Ampr f1 ori
pCAT3-Enhancer Vector
(4293bp) ori
Kpn I Sac I Mlu I Nhe I Sma I Xho I BgI II (Hin d III)
5 11 15 21 28 32 36 53
pCAT3-Control Vector
(4485bp) ori

CAT
1677 Sal I 1671 Bam H I
SV40 Promoter (Hin d III) 245
Kpn I Sac I Mlu I Nhe I Xma I Xho I BgI II
5 11 15 21 28 32 36
1485 Sal I 1479 Bam H I
CAT
SV40 Enhancer
SV40 Enhancer
Hpa I 1131
Xba I 971 Hpa I 1323
Xba I 1163
Figure 6.6. Circle maps of the pCAT3 Vectors.
44
Transfection Guide
Chapter 6
Mammalian Expression Vectors

The heterologous expression of proteins in mammalian cells has become an essential technique to study the physiological function of a protein or the effect of post-translational modications. Promega offers several vectors that can be used for expressing proteins in mammalian cells. Promegas mammalian expression vectors contain the highly active simian virus 40 (SV40) or cytomegalovirus (CMV) promoter and enhancer elements. The pSI, pCI and pCI-neo Mammalian Expression Vectors, and the pTARGET Mammalian Expression Vector System are designed to promote constitutive expression of cloned DNA inserts in mammalian cells (Figure 6.8). Inclusion of the neomycin phos-
photransferase marker in the pCI-neo and pTARGET Vectors allows selection of stably transfected mammalian cells in the presence of the antibiotic G-418. We have improved vector design features, such as intron and polyadenylation regions, which provide enhanced RNA stability and subsequent translation. To aid cDNA subcloning, restriction sites in the multiple cloning regions are compatible with the Universal RiboClone cDNA Synthesis System. For more information on Promegas Mammalian Expression Vectors, please request Technical Bulletins #TB206 (pSI and pCI Vectors), #TB215 (pCI-neo Vector) or Technical Manual #TM044 (pTARGET Vector).
Bgl II
Bgl II
ori SV40 Early Enhancer/Promoter ori
Sgf I
I-Ppo I T7 Nhe I Xho I Eco R I Mlu I Xba I Sal I Acc I Sma I Not I T3
pSI Vector
Ampr
Intron SV40 Late poly(A)
Afl II
(3634 bp)
f1 ori
Bam H I
SV40 Enhancer/ Early Promoter Neo
Bam H I
Bgl II
ori ori CMV I.E. Enhancer/Promoter
Bgl II 5665 Sgf I 664
lacZ
I-Ppo I 851
CMV Enhancer/Promoter
T7
Afl II Pst I
T7 Nhe I Xho I Eco R I Mlu I Kpn I Xba I Sal I Acc I Sma I Bst Z I Not I
1250 1256 1264 1270 1276 1293 1301 1303 1304 1311 1318
678 684 689 695 707 713 714 720 724 724
1085 1091 1096 1102 1114 1120 1121 1127 1131
Afl II Pst I
CMV I.E. Enhancer/Promoter Intron Amp

r
T7 Nhe I Xho I Eco R I Mlu I Xba I Sal I Acc I Sma I Bst Z I Not I
Synthetic poly(A)
Ampr
pCI-neo Vector
(5474bp)
SV40 Late poly(A) f1 ori
Intron
T
pCI Vector
(4008 bp)
Ampr
Intron SV40 Late poly(A)
Afl II
f1 ori
Bam H I
1052 1058 1063 1069 1079 1081 1087 1088 1094 1098 1098
pTARGET Vector
(5670 bp)
SV40 Late poly (A)
Eco R I Bam H I Nhe I Xho I Mlu I T overhang Sma I Kpn I Sal I Acc I Not I Eco R I lacZ
Figure 6.8. Circle maps of the pSI, pCI, pCI-neo and pTARGET Vectors.
Synthetic poly(A)
Neo
f1 ori SV40 Enhancer/ EarlyPromoter
Transfection Guide
45
Chapter 6
Notes
46
Transfection Guide
Cited References
R E F E R E N C E S
1. 2. 3. 4. 5. 6. 7.
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R E F E R E N C E S
67. Loefer, J.-P. and Behr, J.-P. (1993) Meth. Enzymol. 217, 599. 68. Capaccioli, S. et al. (1993) Biochem. Biophys. Res. Comm. 197 (2), 818. 69. Nemoto, Y. et al. (1996) J. Biol. Chem. 271, 13542. 70. Gaiddon, C. et al. (1994) J. Biol. Chem. 269 (36), 22663. 71. Ichijo, H. et al. (1997) Science 275, 90. 72. Dhr, O. et al. (1997) Mol. Pharmacol. 51, 703. 73. Lesch, K.-P. et al. (1996) Science 274, 1527. 74. Demeneix, B.A. et al. (1994) BioTech. 16 (3), 496. 75. Rosoff, M.L., Wei, J. and Nathanson, N.M. (1996) Proc. Natl. Acad. Sci. USA 93, 14889. 76. Boukhnikachvili, T. et al. (1997) FEBS Lett. 409 (2), 188. 77. Loyter, A., Scangos, G.A. and Ruddle, F.H. (1982) Proc. Natl. Acad. Sci. USA 79, 422. 78. Frost, E. and Williams, J. (1978) Virology 91, 39. 79. Lopata, M.A., Cleveland, D.W. and Sollner-Webb, B. (1984) Nucl. Acids Res. 12, 5707. 80. Lowy, D.R., Rands, E. and Scolnick, E.M. (1978) J. Virology 26, 291. 81. Lewis, W.H. et al. (1980) Somat. Cell Genet. 6, 333. 82. Luthman, H. and Magnusson, G. (1983) Nucl. Acids Res. 11, 1295. 83. Gorman, C.M., Howard, B.H. and Reeves, R. (1983) Nucl. Acids Res. 11, 7631. 84. Brash, D.E. et al. (1987) Mol. Cell Biol. 7, 2031. 85. Wilson, S.P., and Smith, A.L. (1996) Anal. Biochem. 246, 148. 86. Al Molish, M.I. and Dubes, G.R. (1973) J. Gen. Virol. 18, 189. 87. Freshney, R.I., (1987) Culture of Animal Cells A.R. Liss, Inc., NY. 88. Alam, J. and Cook, J.L. (1990) Anal. Biochem. 188, 245.
89. Rosenthal, N. (1987) Meth. Enzymol. 152, 704. 90. Groskreutz, D. and Schenborn, E. (1996) Reporter Systems, In: Recombinant Proteins: Detection and Isolation. Tuan, R., ed., Humana Press, Clifton, NJ. 91. Wood, K.V. (1995) Curr. Opin. Biotech. 6, 50. 92. Hollon, T. and Yoshimura, F. K. (1989) Anal. Biochem. 182, 411. 93. DeWet, J.R. et al. (1985) Proc. Natl. Acad. Sci. USA 82, 7870. 94. DeWet, J.R. et al. (1987) Cell. Biol. 7, 725. 95. Thompson, J.F. et al. (1991) Gene 103, 171. 96. Pazzagli, M. et al. (1992) Anal. Biochem. 204, 315. 97. Wood, K.V. (1991) In: Bioluminescence and Chemiluminescence: Current Status. Stanley, P.E. and Kricka, L.J., eds., John Wiley and Sons, NY. 98. Matthews, J.C.et al. (1977) Biochemistry 16, 85. 99. Alton, N.K. and Vapnek, D. (1979) Nature 282, 864. 100. Leslie, A.G.W. et al. (1988) Proc. Natl. Acad. Sci. USA 85, 4133. 101. Seed, B. and Sheen, J-Y. (1988) Gene 67, 271. 102. Neumann, J.R. et al. (1987) BioTechniques 5, 444. 103. Young, D.C. (1993) Anal. Biochem. 215, 24. 104. Groskreutz, D.J. et al. (1995) Promega Notes 50, 2. 105. Lorenz, W.W. et al. (1991) Proc. Natl. Acad. Sci. USA 88, 4438. 106. Farr, A. and Roman, A. (1991) Nucl. Acids Res. 20, 920. 107. Groskreutz, D.J. et al. (1996) Promega Notes 55, 2. 108. Shaw, W.V. (1975) Meth. Enzymol. 43, 737. 109. Cullen, B. et al. (1987) Meth. Enzymol. 152, 687. 110. Hall, C.V. et al. (1983) J. Molec. Applied Gen. 2, 101. 111. Silhavy, T. et al. (1972) In: Experiments in Molecular Genetics, Miller, J.H., ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
48
Transfection Guide
Appendix A
References Using Promega Transfection Reagents

Tfx Reagents for the Transfection of Eukaryotic Cells
1. Fearon, I.M., et al. (1997) Hypoxia inhibits the recombinant 1C subunit of the human cardiac L-type Ca2+ channel. J. Physiol. 500.3, 551-556. Systems Used: Tfx-50 Reagent. Summary: The reagent was used to transfect 293 Cells. 2. Gobin, S.J.P. et al. (1997) Site is crucial for two routes of IFN-induced MHC Class I transactivation: The ISRE-mediated route and a novel pathway involving CIITA. Immunity 6, 601. Systems Used: Tfx-50 Reagent; pGL3 Basic Vector; pGL3 Enhancer Vector; Universal RiboClone cDNA Synthesis System Summary: The Tfx-50 Reagent was used to transfect K562 cells. Cells were assayed for expression of a surface protein by FACS analysis. 3. Grndemann, D. et al. (1997) Primary structure and functional expression of the apical organic cation transporter from kidney epithelial LLC-PK1 cells. J. Biol. Chem. 272, 10408. System Used: Tfx-50 Reagent. Summary: The reagent was used to produce transiently transfected LLC-PK1 and 293 cells as well as stably transfected 293 cells. 4. Ichijo, H. et al. (1997) Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science 275, 90. Systems Used: Tfx-50 Reagent; Transfectam Reagent. Summary: Tfx-50 Reagent was used to produce transient transfectants of human 293 cells. Eight hours post-transfection, the cells were treated with apoptotic agents for 16 hours and assayed. 5. Inui, T. et al. (1997) Cathepsin K antisense oligodeoxynucleotide inhibits osteoclastic bone resorption. J. Biol. Chem. 272, 8109. System Used: Tfx-50 Reagent. Summary: The reagent was used to transiently transfect osteoclasts with antisense oligonucleotides. 6. Keogh, M.-C. et al. (1997) High efciency reporter gene transfection of vascular tissue in vitro and in vivo using a cationic lipid-DNA complex. Gene Therapy, 4, 162. System Used: Tfx-50 Reagent. Summary: The reagent was used to transfect pGL3 Vector into HepG2, HUVEC, and human, rat and rabbit primary cells. The optimized conditions were used to transfect rabbit arteries in vivo and human arteries in vitro.
7.
Ko, B.C.B. et al. (1997) Identication and characterization of multiple osmotic response sequences in the human aldose reductase gene. J. Biol. Chem. 272, 16431. Systems Used: Tfx-50; pGL3 Basic Vector; pGL3 Promoter Vector. Summary: Studies were performed in Chang liver cells using 2g of luciferase reporter and 1g of -galactosidase control in serum-free medium. Cells were transfected 1hr at 37C, the medium removed and fresh medium applied. Twenty-four hours later the cells were assayed.
8.
Nishitoh, H. et al. (1996) Identication of type I and type II serine/threonine kinase receptors for growth/differentiation factor-5. J. Biol. Chem. 271, 21345. System Used: Tfx-50 Reagent. Summary: The reagent was used to transiently transfect COS-1 cells and R mutant Mv1Lu cells.
9.
Nork, T.M. et al. (1997) p53 regulates apoptosis in human retinoblastoma. Arch. Opthamol. 115, 213. System Used: Tfx-50 Reagent. Summary: The reagent was used to transfect WERIRb1 human retinoblastoma tissue culture cells.
10. Rodriquez-Viciana, P. et al. (1997) Role of phosphoinositide 3-OH kinase in cell transformation and control of the actin cytoskeleton by Ras. Cell 89, 457. System Used: Tfx-50 Reagent Summary: NIH/3T3 cells were stably transfected with either puromycin or G418 selection. Cells were assayed 2 weeks after transfection and selected for foci formation in soft agar. 11. Schenborn, E. and Goiffon, V. (1997) Transfection of insect cells with Tfx-20 Reagent. Promega Notes 63, 13. 12. Schenborn, E., Oler, J. and Goiffon, V. (1996) A Trio of Tfx Transfection Reagents for Eukaryotic Cells. Promega Notes 59, 24. 13. Urban, R.J. and Bodenburg, Y. (1996) Transcriptional activation of the porcine P450 11A insulin-like growth factor response element in MCF-7 breast cancer cells. J. Biol. Chem. 271, 31695. System Used: Tfx-50 Reagent. Summary: The reagent was used to transiently transfect MCF-7 cells. 14. Urban, R.J., Nagamani, M. and Bodenburg, Y. (1996) Tumor necrosis factor inhibits transcriptional activity of the porcine P45011A insulin-like growth factor response element. J. Biol. Chem. 271, 31699. System Used: Tfx-50 Reagent Summary: The reagent was used to transiently transfect two variants of the NIH/3T3 cells, NWTb3 and KR1.
Transfection Guide
49
Appendix A
15. Velcich, A. et al. (1997) Organization and regulatory aspects of the human intestinal mucin gene (MUC2) locus. J. Biol. Chem. 272, 7968. Systems Used: Tfx-50 Reagent; pGL2 Basic Vector; pGL2 Enhancer Vector; pGL2 Promoter Vector. Summary: Studies were performed in the intestinal cell lines, HT29 and LS174T, and the HeLa cell line. The Tfx-50 reagent was used to transfect the intestinal cell lines.
6.
Ichijo, H. et al. (1997) Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science 275, 90. Systems Used: Transfectam Reagent; Tfx-50 Reagent. Summary: Transfectam Reagent was used to produce stable transfectants of Mv1LU mink lung epithelial cells using hygromycin selection.
7.
TransFast Reagent
1. Schenborn, E., Goiffon, V. and Oler, J. (1998) An efcient new transfection reagent for eukaryotic cells: TransFast Reagent. Promega Notes 65, 2.
Ito, M., Jameson, J.L. and Ito, M. (1997) Molecular basis of autosomal dominant neurohypophyseal diabetes insipidus: Cellular toxicity caused by the accumulation of mutant vasopressin presursors within the endoplasmic reticulum. J. Clin. Invest. 99, 1897. Systems Used: Transfectam Reagent; CAT Assay System. Summary: Stable transfectants of Neuro2A cells were generated in G418-containing media. The stable transfectants were assayed for the production of arginine vasopressin and CAT enzyme.
Transfectam Reagent for the Transfection of Eukaryotic Cells

1. Carey, D.J., Bendt, K.M. and Stahl, R.C. (1996) The cytoplasmic domain of syndecan-1 is required for cytoskeleton association but not detergent insolubility. Identication of essential cytoplasmic domain residues. J. Biol. Chem. 271, 15253. Summary: The reagent was used to produce stably transfected Schwann cells. 2. Fisher, E.A. et al. (1997) The degradation of apolipoprotein B100 is mediated by the ubiquitinproteasome pathway and involves heat shock protein 70. J. Biol. Chem. 272, 20427. Summary: Transfectam Reagent was used to transfect HepG2 cells. 3. Foley, B.T., Moehring, J.M and Moehring, T.J. (1995) Mutations in the elongation factor 2 gene which confer resistance to diphtheria toxin and Pseudomonas exotoxin A. J. Biol. Chem. 270, 23218. Summary: The reagent was used to produce stably transfected CHO cells. 4. Harada, N. et al. (1997) Human IgGFc binding protein (FcBP) in colonic epithelial cells exhibits mucin-like structure. J. Biol. Chem. 272, 15232. Summary: COS-7 cells were transfected with 10g of DNA and 5l of Transfectam Reagent per 500l of RPMI 1640 media. After 6 hours the medium was changed the cells were cultured for an additional 48 hours prior to assay. CHO cells were plated at 2 x 105 cells per plate 24 hrs prior to transfection as detailed for COS-7 cells. Stably transfected CHO cells were chosen 14 days later after growth in G418. 5. Hinz, M., Moore, M.J. and Bindereif, A. (1996) Domain analysis of human U5 RNA: Cap trimethylation, protein binding and spliceosome assembly. J. Biol. Chem. 271, 19001. Summary: Transfectam Reagent was used to transiently transfect 293 cells. 9. 8.
Jeannin, P. et al. (1997) CD86 (B7-2) on human B cells: A functional role in proliferation and selective differentiation into IgE- and IgG4-producing cells. J. Biol. Chem. 272, 15613. Summary: Studies were performed in COS cells and the cells were assayed 4 days post-transfection. Kennedy, E.D. et al. (1996) Glucose-stimulated insulin secretion correlates with changes in mitochondrial and cytosolic Ca2+ in aequorin-expressing INS-1 cells. J. Clin. Invest. 98, 2524. Summary: INS-1 cells were stably transfected with a neomycin-resistant plasmid and an aequorin expression vector. The cells were assayed by immunocytochemistry, various assays of insulin secretion and Ca2+ channel activity.
10. Kizer, N., Guo, X.-L. and Hruska, K. (1997) Reconstitution of stretch-activated cation channels by expression of the -subunit of the epithelial sodium channel cloned from osteoblasts. Proc. Natl. Acad. Sci. USA 94, 1013. Summary: LM(TK ) mouse cells were stably transfected and selected in media containing hygromycin. The resulting cells were analyzed by Northern blot, Western blot and patch-clamp protocols. 11. Klafki, H.-W. et al. (1996) The carboxyl termini of amyloid peptides 1-40 and 1-42 are generated by distinct -secretase activities. J. Biol. Chem. 271, 28655. Summary: The reagent was used to transiently transfect COS-1 cells.
50
Transfection Guide
Appendix A
12. Lesch,K.-P. et al. (1996) Association of anxiety-related traits with a polymorphism in the serotonin transporter gene regulatory region. Science 274, 1527. Systems Used: Transfectam Reagent; pGL3 Basic Vector; pGL3 Control Vector; pSV--Galactosidase Control Vector. Summary: Studies were performed in human lymphoblasts. For transient expression, lymphoblasts (2 x 105 cells) were exposed for 24 hours to 5g of construct DNA complexed with 5l of Transfectam Reagent in 5ml of RPMI1640. Cells were grown an additional 24 hours, then assayed for luciferase and -galactosidase activity. 13. Nemoto, Y. et al. (1996) Regulatory function of delta/YY-1 on the locus control region-like sequence of mouse glycophorin gene in erythroleukemia cells. J. Biol. Chem. 271, 13542. Summary: Transfectam Reagent was used to transfect MEL Murine Erythroleukemia cells. 14. Nibbs, R.J.B. et al. (1997) Cloning and characterization of a novel murine chemokine receptor, D6:Comparison to three other related macrophage inammatory protein-1 receptors, CCR-1, CCR-3 and CCR-5. J. Biol. Chem. 272, 12495. Summary: The Reagent was used to generate G418-resistant, stably-transfected HEK293 cells. Transfectam 15. Olofsson, A. et al. (1995) Efcient association of an amino-terminally extended form of human latent transforming growth factor- binding protein with the extracellular matrix. J. Biol. Chem. 270, 31294. Summary: Reagent was used to transiently transfect COS-1 cells. Transfectam 16. Rosoff, M.L., Wei, J. and Nathanson, N.M. (1996) Isolation and characterization of the chicken m2 acetylcholine receptor promoter region: Induction of gene transcription by leukemia inhibitory factor and ciliary neurotrophic factor. Proc. Natl. Acad. Sci. USA 93, 14889. Systems Used: Transfectam Reagent; pGL3 Basic Vector; pSV--Galactosidase Control Vector. Summary: Transfectam Reagent was used at 3.5l/g of DNA to transfect primary chicken heart cultures. The DNA/ Transfectam solution was left in contact with the cells for 6-7 hours. Thirty hours post-transfection, the cells were lysed and assayed for luciferase and -galactosidase activity. 17. Smit, M.J. et al. (1996) Two distinct pathways for histamine H2 receptor down-regulation: H2 Leu124Ala receptor mutant provides evidence for a cAMPindependent action of H2 agonists. J. Biol. Chem. 271, 7574. Summary: The reagent was used to stably transfect CHO cells.
18. Venkatakrishnan, G. and Exton, J.H. (1996) Identication of determinants in the -subunit of Gq required for phospholipase C activation. J. Biol. Chem. 271, 5066. Summary: Transfectam Reagent was used to transiently transfect 293 cells. 19. Zhang, L., David, G. and Esko, J.D. (1995) Repetitive Ser-Gly sequences enhance heparan sulfate assembly in proteoglycans. J. Biol. Chem. 270, 27127. Summary: Transfectam Reagent was used to produce stably transfected CHO cells.
Profection Mammalian Transfection SystemCaPO4

1. Behrooz, A. and Ismail-Beigi, F. (1997) Dual control of glut1 glucose transporter gene expression by hypoxia and by inhibition of oxidative phosphorylation. J. Biol. Chem. 272, 5555. Systems Used: ProFection Mammalian Transfection System -CaPO4; Dual-Luciferase Reporter Assay System; pGL2 Basic Vector; pRL-TK Vector; pCAT Control Vector. Summary: The Renilla luciferase activity was used to normalize the rey luciferase activity of transfected Clone 9 cells. 2. Chu, B. et al. (1996) Sequential phosphorylation by mitogen-activated protein kinase and glycogen synthase kinase 3 represses transcriptional activation by heat shock factor-1. J. Biol. Chem. 271, 30847. Summary: NIH/3T3 cells were seeded at 2.5 x 105 cells/100mm dish 24 hours prior to transfection. Cells were assayed 48 hours post-transfection. 3. Hoock, T.C., Peters, L.L. and Lux, S.E. (1997) Isoforms of ankyrin-3 that lack the NH2-terminal repeats associate with mouse macrophage lysosomes. J. Cell Biol. 136, 1059. Summary: The ProFection Mammalian Transfection System was used to transiently transfect COS cells. The DNA precipitates were left in contact with the cells for 16 hours and 48 hours later the cells were analyzed by immunouorescence. 4. Obiri, N.I. et al. (1997) Modulation of interleukin (IL)-13 binding and signaling by the c chain of the IL-2 receptor. J. Biol. Chem. 272, 20251. System Used: ProFection Mammalian Transfection System -CaPO4 Summary: The system was used to produce stable transfectants in the ML-RCC renal carcinoma cell line.
Transfection Guide
51
Appendix A
5.
Walsh, A.A. et al. (1996) Identication of a novel cisacting negative regulatory element affecting expression of the CYP1A1 gene in rat epidermal cells. J. Biol. Chem. 271, 22746. Systems Used: ProFection Mammalian Transfection System -CaPO4; pGL2 Basic Vector; Luciferase Assay System. Summary: Studies were performed in rat epidermal keratinocytes. Cells were grown to 50% conuency in 60mm dishes. Cells were transfected with 15g of plasmid DNA for 24 hours then the media was replaced and the cells assayed 24 hours later.
3.
Zhang, M., Magit, D and Sager, R. (1997) Expression of maspin in prostate cells is regulated by a positive Ets element and a negative hormonal responsive element site recognized by androgen receptor. Proc. Natl. Acad. Sci. USA 94, 5673. Summary: LNCaP human prostate tumor cells, CF3 normal human epithelial cells and 70N mammary epithelial cells were transfected at 75% conuency in a 100mm dish. Cells were assayed 48 hours posttransfection for -gal and CAT activity.
Profection Mammalian Transfection SystemDEAE-Dextran

1. Pierce, R.A. et al. (1996) Monocytic cell type-specic transcriptional induction of collagenase. J. Clin. Invest. 97, 1890. Summary: U-937 cells (1 x 107cells) were transfected in suspension with 10g of DNA and 75g of DEAEDextran for 20 min at room temperature. The cells were divided and allowed to recover overnight. The cells were treated with a reagent and CAT activity assayed 24 hours later. 2. Rohlff, C. et al. (1997) Modulation of transcription factor Sp1 by cAMP-dependent protein kinase. J. Biol. Chem. 272, 21137. Systems Used: ProFection Mammalian Transfection System-DEAE-Dextran; pCAT Basic Vector; pCAT Promoter Vectors, Recombinant Human Sp1. Summary: Reporter studies were performed in HL-60 and HL-60/AR (doxorubicin-resistant isolate) leukemia cells. DNA was transfected by electroporation in the presence of DEAE-Dextran and many details of the transfection are reported. Recombinant Sp1 was in vitro phosphorylated with the cAMP dependent protein kinase catalytic subunit and used in gel shift assays.
References Using Promegas Reporter Assay Systems and Expression Vectors

References using Promegas reporter assay systems and expression vectors are available on the Internet at www.promega.com.
52
Transfection Guide
Appendix B
Transfection Systems
Product TransFast Transfection Reagent Size 1.2mg Cat.# E2431
Reporter Assays and Vectors

Luciferase Reporter Vectors and Assay Systems
Product pRL-TK Vector pRL-CMV Vector pRL-null Vector pRL-SV40 Vector pGL3-Control DNA pGL3-Basic DNA pGL3-Enhancer DNA pGL3-Promoter DNA pGL2-Control DNA pGL2-Basic DNA pGL2-Enhancer DNA pGL2-Promoter DNA Dual-Luciferase Reporter Assay System Luciferase Assay System With Reporter Lysis Buffer Luciferase Assay System Size 20g 20g 20g 20g 20g 20g 20g 20g 20g 20g 20g 20g 100 assays 100 assays 100 assays Cat.# E2241 E2261 E2271 E2231 E1741 E1751 E1771 E1761 E1611 E1641 E1621 E1631 E1910 E4030 E1500
Contains sufcient reagent to transfect 400g of DNA (at a 1:1 TransFast Reagent:DNA ratio).
Product Tfx-10 Reagent Tfx-20 Reagent Tfx-50 Reagent
Size 9.3mg 4.8mg 2.1mg
Cat.# E2381 E2391 E1811
Each system contains sufcient reagent to transfect 200g of DNA (at a 4:1 Tfx Reagent:DNA ratio).
Product Tfx Reagents Transfection Trio
Size 5.4mg
Cat.# E2400
Contains one tube each of Tfx-10, Tfx-20 and Tfx-50 Reagents. Each system contains sufcient reagent to transfect 200g of DNA (at a 4:1 Tfx Reagent:DNA ratio).
Product Transfectam Reagent for the Transfection of Eukaryotic Cells ProFection Mammalian Transfection System Calcium Phosphate ProFection Mammalian Transfection System DEAE-Dextran
Size
Cat.#
1mg 0.5mg
E1231 E1232
CAT Reporter Vectors and Assay Systems

Product pCAT3-Basic Vector pCAT3-Enhancer Vector pCAT3-Promoter Vector pCAT3-Control Vector pCAT-Basic DNA pCAT-Enhancer Vector pCAT-Control DNA pCAT-Promoter DNA CAT Enzyme Assay System With Reporter Lysis Buffer Chloramphenicol Acetyltransferase (CAT) Size 20g 20g 20g 20g 20g 20g 20g 20g Cat.# E1871 E1881 E1861 E1851 E1041 E1021 E1011 E1031 E1000 100u E1051
40 reactions
E1200
40 reactions
E1210
Eukaryotic Expression Vectors

Product pCI-neo Mammalian Expression Vector pCI Mammalian Expression Vector pSI Mammalian Expression Vector pTARGET Mammalian Expression Vector Size 20g 20g 20g 20g Cat.# E1841 E1731 E1721 A1410
-Galactosidase Reporter Vectors and Assay Systems

Product pSV--Galactosidase Control Vector -Galactosidase Enzyme Assay System With Reporter Lysis Buffer Reporter Lysis Buffer 5X Size 20g Cat.# E1081
65 assays 30ml
E2000 E3971
continued
Transfection Guide
53
Appendix B
Luminometers
Product Turner Designs Luminometer Model TD-20/20 Genetic Reporter Instrumentation Package for Stabilized Assays Turner Designs Luminometer Model TD-20/20 Genetic Reporter Instrumentation Package for Stabilized Assays, with Printer Turner Designs Luminometer Model TD-20/20 Genetic Reporter Instrumentation Package for Stabilized Assays, with Printer and Dual Auto Injector System Turner Designs Luminometer Model TD-20/20 Genetic Reporter System with Single Auto Injector Turner Designs Luminometer Model TD-20/20 Genetic Reporter System with Dual Auto Injector Cat.#
E2041
E2051
E2061
E2351
E2361
54
Transfection Guide
Appendix B
Notes
Transfection Guide
55
Appendix B
Notes
56
Transfection Guide
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Transfection

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Transfection

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Transfection

Recommended Promega Transfection Reagent for Commonly-Used Cell Lines.

Promegas Transfection, Reporter Assay Family and Eukaryotic Expression Vectors

Reporter Vectors and Assay Systems

Renilla Luciferase Control Vectors

Eukaryotic Expression Vectors

Historical Background. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Transfection Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

References Using Promega Transfection Reagents. . . . . . . . . . . . . . . . . 49

1998 Promega Corporation. All Rights Reserved.

Reporter Gene Plasmid DNA

Cell Lysates Enzyme Activity

Luciferase CAT -Galactosidase

Figure 1.1. Reporter Gene Systems. Transfection Guide 5

Ca++ DNA DNA

O O + N C O C O Cationic Head Group Link Lipid

Figure 1.3. General structure of a synthetic cationic lipid.

Figure 1.4. Structure of DOPE (L-dioleoyl phosphatidylethanolamine).

Preparation of DNA for Transfection

Plasmid Preparation Protocol

Cesium Chloride Equilibrium Gradient

Anion Exchange Chromatography

Preparation of Cells for Transfection

Transient Expression vs. Stable Transfection

Assay for Gene Expression

Apply Selective Pressure

Figure 2.1. Schematic representations of stable and transient transfections.

Composition of Buffers and Solutions

NaCl KCl Na2HPO4 KH2PO4

Figure 3.1. Structure of Transfectam Reagent.

Liposome Based Transfection Reagents

Advantages of Using Cationic Lipid Reagents for Transfection

Factors Inuencing Transfection Efciency

Relative Light Units (x 10-4)

10 5 0 125 250 500 1,000 125 250 500 1,000 1,000

ng DNA Tfx-10 Tfx-20 Tfx-50 Tfx-10

Relative Light Units (x 10-2)

ng DNA Tfx-20 Tfx-50

Relative Light Units (x 10-4)

Relative Light Units (x 10-2)

40 35 30 25 20 15 10 5 0 125 250 500 1,000 125 250 500 1,000 1,000

ng DNA Tfx-10 Tfx-20 Tfx-50 Tfx-10

ng DNA Tfx-20 Tfx-50

Liposome Based Transfection Protocols TransFast and Tfx Reagents

Relative Light Units/Well

200,000 150,000 100,000 50,000

Cell Line 293

Reagent TransFast Reagent

3:1 (4:1) 1:1

TransFast Reagent TransFast Reagent TransFast Reagent Tfx-20

1:1 (2:1) 1:1

TransFast Reagent SF9 Insect Tfx-20

Day One Plate cells, resuspend and freeze liposome reagent.

1. Add liposome reagent to the DNA/Medium mixture and vortex briefly.

Day Two Dilute DNA in medium.

2. Incubate 10-15 minutes.

Remove the growth medium from the cells.

3. Add DNA/ liposome complex to cells.

Perform the desired assay.

Size of Plate 24 well 96 well 12 well 6 well 35mm 60mm 100mm

Relative Areab 1X 0.2 X 2X 5X 4.2 X 11 X 29 X

This information was calculated for Corning culture dishes.

Preparation of Liposome Reagent Stock Solution