Studying MHC function by

reverse genetics
IMM 3031
Friday April 15th 2016
2pm lect.
Theatre M2
A/Prof Robyn Slattery
robyn.slattery@monash.edu

Learning objectives
Be able to describe the basics of genetic
engineering approaches including:
Transgenic manipulation
Homologous recombination to generate knockout, knock-in and tissue specific modifications
Using ES cells, traditional gene targeting and cre-lox
Using CRISPR technology

Describe examples of each of these

manipulations and how these have been
used in immunological research.
2

Methods of finding or
making mutants
1. Screen organism for naturally occurring and
interesting mutations -phenotypes
2. Treat organisms with (UV light, chemicals,
transgenes) and then screen for interesting
mutants -phenotypes
3. Generate transgenics
4. Target mutations to specific genes by
homologous recombination
a) cre/lox technology (1993-2013)
b) CRISPR technology (since 2013)
3

Methods of finding or
making mutants
1. Screen organism for naturally occurring and
interesting mutations -phenotypes
2. Treat organisms with (UV light, chemicals,
transgenes) and then screen for interesting
mutants -phenotypes
3. Generate transgenics
4. Target mutations to specific genes by
homologous recombination
a) cre/lox technology (1993-2013)
b) CRISPR technology (since 2013)
4

Making transgenic mice

Exon

Intron
Gene
or
cDNA

5 regulatory region!
(promoter region)!
- specificity!

Coding region!
- governs what the gene!
product will be!
5

Construct
transgene
holding pipette!

X!
1-cell embryo!
at pronuclear stage!
!

oviduct transfer!
X

Screen litters!
for transgenic!
mice!
7

jellyfish

Adding fluorescent protein

plant

mice
fish

Slattery et al Nature345, 724 - 726 (1990);

Prevention of diabetes in non-obese diabetic I-Ak transgenic mice

"

Normal MHC class II

Diabetes"

No diabetes"

In Summary
Transgenic mice give us an extra gene i.e.
gain of function
How do we develop other mutants?
Complete loss of gene function (knock-outs)
Change of gene function (knock-ins)
regulated loss of gene function (tissue-specific
knock-outs)

10

Methods of finding or
making mutants
1. Screen organism for naturally occurring and
interesting mutations -phenotypes
2. Treat organisms with (UV light, chemicals,
transgenes) and then screen for interesting
mutants -phenotypes
3. Generate transgenics
4. Target mutations to specific genes by
homologous recombination
a) ES cells and cre/lox technology (1993-2013)
b) CRISPR technology (since 2013)
11

Using homologous recombination in

ES cells to generate:
Gene knock-outs
Gene knock ins
Tissue specific gene knock-outs

12

Genetically modified ES cells give rise to genetically

modified mice
transgene transfer by transfection

Embryonic stem
cells

Inner cell mass

Select and transfer ES cells

To blastocyst

Blastocyst

oviduct transfer!
Look for chimeric mice

Breed mice

Screen litters!
for transgenic!
mice!
13

homologous recombination to
generateknock-out mice
Process which basically replaces endogenous (functional) gene with in
vitro manipulated (non-functional) gene.

Gene to be targeted!

Gangcyclovir
HSV-TK
Toxic

Neo R

Targeting vector!

selectable markers!

Neo R= Neomycin Resistance Gene

HSV-TK= Herpes Simplex Virus Thymidine Kinase

14

Use of knock-out mice to prove that MHC

class I is required for the generation of
CD8 T cells and the development of
diabetes
Diabetes. 1994 Mar;43(3):500-4.

2M-deficient NOD mice do not develop

insulitis or diabetes.



Wicker LS, Leiter EH, Todd JA, Renjilian RJ, Peterson E,

Fischer PA, Podolin PL, Zijlstra M, Jaenisch R, Peterson LB.

15

homologous recombination to
generate knock-in mice
Concept can be extended to introduce defined changes to test specific
feature or function.

Gene targeting vector!

Altered nucleotide(s)!

16

The use of
tissue-specific knock out mice
to determine the role to MHC molecules have in T1D

CD8"

APC"
-cell

CD4"

CD8"

tissue specific knockout of MHC class I

expression from Islet beta cells"

CD8"

APC"
-cell

CD4"

CD8"

tissue-specific knock-out mice

Gene to be targeted!
Gene of interest!
Flanked by lox sites!

Targeting vector!

Endogenous gene flanked by lox sites

Cre mediated recombination at lox sites"

cre"

In vivo cre mediated recombination at

lox sites"
X"
Gene flanked by lox"

cre"

Tissue specific cre"

issue-specific knock-out mouse

21

Islets from class-I--bald NOD and controls

2M+/-

CD4"

CD8"

Macrophage"

2Mloxcre-
2Mloxcre+

Islets from class-I--bald NOD and controls

2M+/-

2Mloxcre-
2Mloxcre+

CD4"

CD8"

Macrophage"

NOTE:
CD8 T cells are still
present in the islet
milieu despite the
lack of MHC class I
expression on islet
beta cells

Cumulative diabetes "

Incidence (%)"

Reduced incidence of diabetes in

class I beta baldNOD mice
floxed2Ma"
HIPcre-"
N=27"

20"
15"
10"
5"

floxed2Ma"
HIPcre+"
n=39"

0"

2M-/-"
HIPcre+"

100" 120" 140" 160" 180" 200" 220" 240"

Age (days)"

n=17"

Use of tissue-specific knock-out mice

demonstrated MHC class I is required on
the target beta cells for CD8 T cells to kill
them and cause T1D
Proc Natl Acad Sci U S A. 2003 May 27;100(11):6688-93. Epub
2003 May 15.

Beta cell MHC class I is a late requirement for diabetes.



Hamilton-Williams EE, Palmer SE, Charlton B, Slattery RM.

25

Class I removed from APCs"

CD8"

APC"
-cell

CD4"

CD8"

Can remove class I from

many different subsets
APCs"
CD8"

CD8"

beta-cell

dend"

CD8"

CD4"

CD8"

CD4"
m"

B cell"

Can remove class I from

many different subsets
APCs"
CD8"

CD8"

beta-cell

dend"

CD8"

CD4"

CD8"

CD4"
m"

B cell"

Can remove class I from

many different subsets
APCs"
CD8"

CD8"

beta-cell

dend"

CD8"

CD4"

CD8"

CD4"
m"

B cell"

Using CRISPR-Cas technology to make

genetic modifications in vitro and in
vivo"

30

CRISPR-cas
its origin and constituents
CRISPR = clustered, regularly interspaced, short
palindromic repeats
CRISPR is a genomic locus in some bacteria and
archaea that funtions as an adaptive immune system
against invading phage or plasmids
Encodes an endonuclease
Stores snippets of foreign sequence
Transcribed into RNAs that guide the endonuclease by base
complementarity to cleave foreign nucleic acids at specific sequences

Type II CRISPR systems

Encodes endonuclease Cas9
Has guide RNA to direct Cas9 to virtually any desired
genomic sequence

CRISPR-cas how does this bacterial

immune system work?
Sontheimer et al, 2010, Nature, 438: 45-46.
Jenkins, J., Biotechniques, July 2012

CRISPR-cas
its origin and constituents
CRISPR = clustered, regularly interspaced, short
palindromic repeats
CRISPR is a genomic locus in some bacteria and
archaea that funtions as an adaptive immune system
against invading phage or plasmids
Encodes an endonuclease
Stores snippets of foreign sequence
Transcribed into RNAs that guide the endonuclease by base
complementarity to cleave foreign nucleic acids at specific sequences

Type II CRISPR systems

Encodes endonuclease Cas9
Has guide RNA to direct Cas9 to virtually any desired
genomic sequence

CRISPR-cas
Targeted genome editing
Targeted genome editing

Genomic DNA

Site-specific dsDNA
break

Genome specific tracrRNA-crRNA

chimera

Cleaving single-stranded
RNA-guided Cas9 protein

Genome specific
crRNA sequence

Matching genomic
sequence

Sontheimer et al, 2010, Nature, 438: 45-46. Jenkins, J., Biotechniques,

July 2012

Modification of targeted genome

CRISPR-cas
a two component system
2. Plasmid encoding Cas9
transfected into cell

1.
(critical residues for specificity in red, seed
sequence) linked to tracrRNA

- interacts with Cas9 protein

Schematic of a CRISPR/Cas-targeted double-strand break

CRISPR-cas
genome editing options
Double stranded break

Donor template present?

no

yes

yes
Precise DNA modification via
homology-directed repair

Gene knockout via nonhomologous end joining

NHEJ:

HDR:

HDR:
A

or
ssOligo donor

Error-prone

plasmid donor

Error-free

x
Gene disruption
(knockout)

A
T

DNA insertion
Gene correction
Precise DNA modification
or deletion

CRISPR-cas
genome editing options
Double stranded break

Donor template present?

no

yes

yes
Precise DNA modification via
homology-directed repair

Gene knockout via nonhomologous end joining

NHEJ:

HDR:

HDR:
A

or
ssOligo donor

Error-prone

plasmid donor

Error-free

x
Gene disruption
(knockout)

A
T

DNA insertion
Gene correction
Precise DNA modification
or deletion

NHEJ

CRISPR-cas
genome editing options
Double stranded break

Donor template present?

no

yes

yes
Precise DNA modification via
homology-directed repair

Gene knockout via nonhomologous end joining

NHEJ:

HDR:

HDR:
A

or
ssOligo donor

Error-prone

plasmid donor

Error-free

x
Gene disruption
(knockout)

A
T

DNA insertion
Gene correction
Precise DNA modification
or deletion

HDR

Repairing DSBs

Non-homologous end-joining (NHEJ)

Sticks ends back together

Error-prone (can create knock out)

Homology directed repair (HDR)

>100X more efficient for making mutants than traditional gene targeting
due to:

DS breaks

Direct zygote injection (no need for chimeras)

High fidelity

Requires longer regions of homology (usually >500 bp)

CRISPR-Cas9 can also be

used to activate and repress
genes

Using a catalytically deadCas9 CRISPRCas9 can also be used to activate and

repress genes
2. Plasmid encoding dCas9
transfected into cell

dCas9 Binds but does not cut

1.
(critical residues for specificity in red, seed
sequence) linked to tracrRNA

- interacts with Cas9 protein

Schematic of a CRISPR/Cas-targeted double-strand break

Activation of genes using

CRISPR-cas9

Repression of genes using

CRISPR-cas9

CRISPR-cas
genome editing can be performed for either in
vitro or in vivo studies
CRISPR Workflows

Gene Synthesis
Workflow
(eg gene knock-in or modification)

FACS
In Vitro studies

Transfect cells

Select GFP+
cells

VALIDATION
Cel-1 assay
Mutation
detection
assay

Single cell
proliferation

Serial dilution

ECACC Cells

PCR reagents

Media & supplements

Media & Supplements

Oligonucleotides

Plates, tips, buffers

Escort TM transfection

GenElute

stable cells
Freezing
reagents

Media &
supplements
Plates, tips, buffers

Cryo vials

TM

Plates, tips, buffers

0.5

Weeks

0.5

1.0

2.0

4.0

In Vivo studies

CRISPR

4.0

~ 12 weeks
PCR reagents

Isolate One-cell
embryo

Extract-N-Amp

Cryopreservan
t

Oligonucleotides

Microinject
into
nucleus
CRISPR mRNA

Weeks

2.0

TM

Embryo culture media

1.0

Select and transfer CRISPR

modified embryos
to foster mother

2.0

Genotype founder pups

for targeted gene manipulation

11.0

~ 16 weeks

CRISPR-cas
its potential over the next few years?
Can be used for in vitro disease modeling in
combination with differentiated tissue derived
from induced stem cells
to study the significance of specific genetic
contributors to phenotypic features
to modify certain features of the tissue prior to
replacement therapy
e.g. beta cells (genetically engineered to be resistant to
recurrent autoimmune attack) for transplantation into
diabetic patients
Questiondo you think beta cells would survive in
transplanted recipients if engineered to lack MHC class I?

CRISPR-cas
Can achieve everything older gene targeting
methods can
Faster
cheaper
More efficient
Can target multiple genomic sites at once

CRISPR-cas
applications in past 2 years

Rice Plants

C. elegans

Fish

Rat

Tobacco Plants

Drosophila
Bacteria

AND
non-human primates
Human cells

Yeast

Mouse
Arabidopsis

 https://www.youtube.com/watch?
v=2pp17E4E-O8

Summary of this lecture

We can develop directed mutants that cause

Complete loss of gene func:on (knock-outs)

Change of gene func:on (knock-ins)
regulated loss of gene func:on (:ssue-specic knock-outs)

This can be done to generate modied plants and

animals using either tradi:onal gene targe:ng tools
ES (embryonic stem cells) and cre-lox technology
(1993-2013)

Since 2013 this can also be done using CRISPR/Cas

technology
There are many advantages to this new approach
especially its applica:on to regenera:ve medicine

51

Reading
See Janeway 8th ed (Immunologists toolbox)
Hamilton Williams et al (2003) Beta cell MHC
class I is a late requirement for diabetes PNAS
Vol100(11):6688-6693
deJersey et al (2006) Beta cells cannot directly
prime diabetogenic CD8 T cells in NOD mice
PNAS Vol104(4):1295-300
Gaj T et al (2013) ZFN, TALEN and CRISPR/Casbased methods for genome engineering Trends in
Biotchnology Vol 31(7): pp397-404
Bondy-Donomy & Davidson (2014) To acquire or
resist: the complex biological effects of CRISPRCas systems Trends in Microbiology Vol 22(4):
pp218-225
52