Problem Set 4 - LS4 Problem Set 4, 2010 Transgenics and...

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LS4 Problem Set 4, 2010 Transgenics and homologous recombination Corresponding Lectures: November 17, 19 (maybe the beginning of Nov 22) Corresponding Reading: Hartwell et al., 744-745, 851-852, 858-859 Corresponding Quiz: November 29-December 3 Problem 1. You decide to make a knock out of your favorite gene in mice. You design a transgene for the knockout, but your colleague tells you that it won’t work. (R of H = region of homology) What is wrong with this transgene, and how would you fix it? Problem 2. L would like to make a knock-out mouse but isn’t really sure how to do it properly. He decides to make three different targeting transgenes, hoping that at least one will work. He makes the 3 transgenes below and transforms them into ES cells, then adds neomycin and gancyclovir. For each transgene, describe whether a cell will live or die if a ) the transgene does not integrate, b ) the entire transgene integrates randomly into the ES celll genome, c ) homologous recombination occurs properly. In each case that a cell dies, describe which compound or compounds will kill it. (ROH=region of homology, NeoR=neomycin resistance gene, TK=thymidine kinase)
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Problem 3. G has successfully knocked out Her Favorite Gene (HFG) in ES cells and made one chimeric mouse with those cells. Unfortunately, she was only able to get one female mouse pup heterozygous for the HFG knockout from the chimera before it died. Ultimately, G wants to see the homozygous mutant phenotype for HFG. Does she have to make a new chimera? Or is there an easier way to obtain a homozygous mutant? Problem 4. W has successfully knocked out Gene B in ES cells and injected those cells into a host blastocyst. From these experiments he has obtained two chimeric mice. He has also invented a technique for estimating the chimerism of the somatic cells and of the pre-meiotic germline cells. Using this technique he has discovered that Mouse 1 is a male; 40% of his somatic cells and 50% of his germline cells are derived from the ES cells. Mouse 2 is a female; 60% of her somatic cells and 30% of her germline cells are derived from ES cells. A. If he mates the chimeric male to a wild-type female, what percentage of the progeny will be fully wild-type, what percentage will be heterozygous for the knock-out gene, and what percentage will be homozygous for the knockout gene? B. If he mates the chimeric female to a wild-type male, what percentage of the progeny will be fully wild-type, what percentage will be heterozygous for the knock-out gene, and what percentage will be homozygous for the knockout gene? C.
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Problem Set 4 - LS4 Problem Set 4, 2010 Transgenics and...

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