BIMM 110 Section 4 40

DAYS TIL
FINAL

ROR1 Expression in Cancer Cell Lines as a

model of Cell Line and Animal Model Studies

...and then some other unrelated stuff.

George Chen
April 27, 2009
gtchen@ucsd.edu
www.scribd.com/g_chen
 Announcements
● Sorry about missing my OH, will reschedule (see site)
● Midterm next Tuesday
● Last names A-K Solis 104
● Last names L-Z Pepper Canyon 106
● Bring ID and Pen
● Review session: Sunday 8-9:20 pm PCYN 106
● No section next week
● Twin studies guest lecture will be covered on midterm
● Will try to make a problem set for lectures 9 & 10, check
website
 Background
● ROR1 is an oncofetal protein that is expressed
only in early development and in some cancer
cells
● Overexpression of ROR1 leads to aggressive
tumor growth
● Silencing of ROR1 causes poor viability and
slow growth in vivo
 Study Goals
● Transfect ROR1 into a non-expressing cancer
cell line and observe growth
● Detect ROR1 expression in mice cancers to use
as an animal model
 Cell Culture
● Immortal cell lines are
derived from cancers
● Can be stored in liquid
nitrogen indefinitely
 Cell Culture
● Cells are cultured in cell growth media that is
supplemented by serum (typically Fetal Bovine
Serum), and usually some antibiotics.
● Typical cell culture media are RPMI and DMEM
1. Stable cell line ROR1 expression
● Culture MCF7 and SKBR3, two breast cancer
cell lines that do not express ROR1
● Transfect with ROR1 plasmid grown from
bacteria
● Select for ROR1 transfected cells using drug
selection
● Detect ROR1 expression using flow cytometry
 Transfection
● Chemical transfection
● Electroporation
● Transient vs. Stable transfection
 Transfection efficiency
● A measure of how many cells took up the
inserted plasmid
● Can usually be tested with GFP

GFP → ← dsRed
Merge → ← Normal
 Flow cytometry
● A technique for quickly sorting, counting, or
observing cells or other small particles, ie.
proteins

Control ROR1 ab

EW36 (ROR1 +) MCF 7 (ROR1 -)

 2. Mouse models
● Check for ROR1 expression of mouse cancer
cells
● Previously, bred knockout mice to watch for
ROR1 activity on a MDA-MB-231 human breast
cancer tumor.
● Used “nude mice” - have no adaptive immune
system
 Knock out/Knock in
● Use homologous recombination
● Neo - Neomyocin resistance
● HSV-tk – Gangcyclovir sensitivity
● Conditional knockout (avoid embryonic lethality)
● LoxP attached to gene of interest, transfected
● Cre recombinase can be used to selectively
express the gene by binding to LoxP
 Genetic Map
● Based upon recombination frequency
● 1 cM = ~1 million bp = 1% recombination
● Measure recombination in a heterzygous x
homozygous recessive cross
LOD (Logarithm of Odds) – Z score
Likelihood of linked
LOD=log 10
Likelihood of unlinked
● A statistical estimate of whether two loci are
likely to be near each other
●
θ unlinked = 0.5
●
θ linked = between 0 and 0.5
●
Z score is determined by θ , the θ value that
gives the highest LOD score gives the best
estimate
 Calculating LOD
●
Multiple Z scores are calculated from varying θ
values
● Ideal to have a Z score of 3 or more. If -2, then
there is no linkage
●
LOD scores for a given θ in different pedigrees
are additive
● This can cause the overall Z score to go up/down
 Physical mapping
● Use vectors from overlapping clones
● Functional gene cloning
● Known protein
● Deduce sequence, identify chromosome region
● Positional
● Unknown protein
 SNPs
● Linked – no effect on protein
production/function
● Causative – inside gene of interest, no effect
● Noncoding – changes amount of protein
produced
● Coding – changes AA sequence

● Useful as a genetic marker