Transgenic & KO mice- Genetics

  • Created by: Sarah
  • Created on: 27-04-13 20:01
What are the applications of KO mice?
model for hum disease, drug assessment, understanding gene reg/control
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What is forward genetics?
From phenotype to gene e.g. by isolating genes via mutagenesis screenings- recessive mut induced chemically in male germ line and breed for 2 generations until homo offspring observed then analyse pheno and clone
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What is reverse genetics?
From gene to phenotype. KO or site directed mutagenesis to investigate the effects on the phenotype.
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What are the 2 ways that transgenic mice are generated?
Pronuclear injection, ES cells
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How is pronuclear injection done?
DNA from cloned gene into male pronucleus & transferred to oviduct. Multiple lines must be established due to variability, southern blot analysis to confirm transgenic status
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How are transgenic mice generated from ES cells?
inner cell mass cells from blastocyte cultured then fused into cloned gene vector, injected into host embryo to form chimeric embryo. Chimeric blastocyte injected into uterus of primed mother, southern blot or PCR analysis.
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What happens when you cross chimeric mice with wild type mice?
All offspring are heterozygous.
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what is positional cloning?
When a gene's proximal chromosome location is identified (but not the function) e.g. linkage analysis identifies candidate region then positional cloning narrows it down to the gene.
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What is SYR region?
testis determination, positional cloning showed its on Y chrom, mice with SYR show sex reversal, ** mice with SYR transgene develop as males, it is mutated in XY females
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Describe step 1) of reverse genetics (KO mice)
Gene of interest must be disrupted e.g. antibiotic resistance gene neo(r) is pliced into cloned gene which gives it neomycin resistance
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step 2)
Disrupted gene becomes part of recombinant DNA molecule with tk gene for herpes virus (this is the targeting vector). This encodes thymidine kinase which phosphorylates ganciclover.if cell expressing tk is grown on ganciclover replic won't take place
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step 3)
Homologous recombo takes place once target vector is transformed into ES cells.
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Step 3) what happens during homologous recombo?
Gene of interest and neomycin resistance gene are transferred from target vector to ES cell chromosome.
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Step 3) where does this recombo occur?
medium containing neomycin and ganciclover so the only cells that survive have the neomycin resistance gene and haven't incorporated the tk gene.
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step 4)
Surviving ES cells are tested using southern blot or PCR to verify KO.
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step 5)
ES cells are injected into a blastocyte which is injected into the uterus of another mouse.
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If KO is present in the germ line then half the offspring of wt mouse and chimeric mouse will show what phenotype?
Half will be Hz for agouti colour
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How do you then establish homo KO mice?
Test DNA in F1 progeny and those containing KO are mated to establish homo progeny
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Other cards in this set

Card 2

Front

What is forward genetics?

Back

From phenotype to gene e.g. by isolating genes via mutagenesis screenings- recessive mut induced chemically in male germ line and breed for 2 generations until homo offspring observed then analyse pheno and clone

Card 3

Front

What is reverse genetics?

Back

Preview of the front of card 3

Card 4

Front

What are the 2 ways that transgenic mice are generated?

Back

Preview of the front of card 4

Card 5

Front

How is pronuclear injection done?

Back

Preview of the front of card 5
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