Bimm 110 Section 4 Slides

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 [email protected] www.pdfcoke.com/g_chen

Announcements ●

Sorry about missing my OH, will reschedule (see site)

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Midterm next Tuesday ●

Last names A-K

Solis 104

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Last names L-Z

Pepper Canyon 106

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Bring ID and Pen

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Review session: Sunday 8-9:20 pm PCYN 106

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No section next week

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Twin studies guest lecture will be covered on midterm

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Will try to make a problem set for lectures 9 & 10, check website

Background ●

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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 ●

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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 ●

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Immortal cell lines are derived from cancers Can be stored in liquid nitrogen indefinitely

Cell Culture ●

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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 ●

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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

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Electroporation

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Transient vs. Stable transfection

Transfection efficiency ●

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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

EW36 (ROR1 +)

ROR1 ab

MCF 7 (ROR1 -)

2. Mouse models ●

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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 ●

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Use homologous recombination ●

Neo - Neomyocin resistance

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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

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1 cM = ~1 million bp = 1% recombination

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Measure recombination in a heterzygous x homozygous recessive cross

LOD (Logarithm of Odds) – Z score

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Likelihood of linked LOD=log 10 Likelihood of unlinked A statistical estimate of whether two loci are likely to be near each other

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θ unlinked = 0.5

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θ linked = between 0 and 0.5

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Z score is determined by θ , the θ value that gives the highest LOD score gives the best estimate

Calculating LOD ●

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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

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Functional gene cloning

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Known protein

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Deduce sequence, identify chromosome region

Positional ●

Unknown protein

SNPs ●

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Linked – no effect on protein production/function Causative – inside gene of interest, no effect Noncoding – changes amount of protein produced

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Coding – changes AA sequence

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Useful as a genetic marker