Lecture13-Functional Genomics_UPDATED Draft

Lecture13-Functional Genomics_UPDATED Draft - Lecture 13 -...

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1 Lecture 13 - Functional Genomics Part III Tuesday Nov 1, 2011 Objective : Know the various reverse genetics methods for knocking out (or knocking down) individual genes and collections of genes on a genome-wide scale Hartwell, pp. 629 (protein detection and tagging) pp. 621-622 (knockout mice)
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2 Experimental strategies in functional genomics (Lectures 11-13) Basic approaches that describe/identify: G e ne  e xpre s s io n pa tte rns P ro te in e xpre s s io n pa tte rns  Pro te in-pro te in inte ra c tio ns   Mutant phenotypes
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3 REVERSE GENETICS: a  po we rful to o l fo r m a nipula ting  a nd e xplo ring  g e ne  func tio n
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4 Mutant Phenotypes: Gene Knockout Methods   Targeted Gene Replacement  RNA Interference Insertional or chemical mutagenesis These methods have been used to different degrees of success in  various model organisms
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5 targets a particular site (gene) in the genome disrupts/replaces the native gene is based on homologous recombination (exploiting the  organism’s own homologous recombination machinery) is now a standard procedure in two model systems: yeast and  mouse Targeted gene replacement - a  po we rful te c hniq ue  tha t a llo ws  yo u to  dis rupt a ny s pe c ific   g e ne  a t will
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6 Homologous recombination in yeast Homologous recombination occurs at high frequency Precise with no gaps and no insertion of new sequences at crossover point Only need 100 bp of sequence homology on either side Yeast chromosome Replacement construct Resulting chromosome selectable marker selectable marker
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Gene Knockouts  have been  e ng ine e re d in e ve ry ye a s t g e ne   (~6,000 g e ne s ) * WHAT C O ULD YO U DO  WITH  S UC H A C O LLEC TIO N?
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8 Knockout Mice: Targeted gene replacement requires a construct with 2 markers “neo” gene confers resistance to neomycin “tk” gene confers sensitivity to ganciclovir (cells die in ganciclovir)
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9 Illustration of how to create a chimeric mouse by mixing together embryonic cells from different parents This mass of cells can be cultured as embryonic stem (ES) cells Is the white mouse genetically related to the chimeric offspring?
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In this example, the ES cells carrying the recombination event come from a non- pigmented mouse. NEXT, the ES cells carrying the gene
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Lecture13-Functional Genomics_UPDATED Draft - Lecture 13 -...

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