Lecture 14 - Methods in Molecular Biology III

Lecture 14 - Methods in Molecular Biology III -...

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Methods in Molecular Biology III Homologous Recombination can also be used in totipotent (embryonic) stem cells (ES cells) of mice G-418 will select for all recombination events With the tk gene, mice are fine in presence of ganciclovir - if the ES cells have taken up more than the desired gene (have take a tk too), they will die in ganc. Only ES that have undergone the appropriate HR will survive the two screens The embryo must be of a mouse with dominant coat colour (black) Blastocyst is transferred to surrogate mother of dominant coat colour -progeny will be chimeric (surrogate, some of mothers cells, some of altered ES) and black (mother’s) Some of the ES cells that were altered may now be in the ES of the chimeric Chimeric is then crossed with a homozygous recessive (black) mouse to see if germ line was attained
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Unformatted text preview: Brown (dominant) babies can be X-/X+ (one X from ES germ line, one from black mouse) or X+/X+: germ line missed Screen for heterozygotes mate heterozygotes screen progeny for homozygotes X-/X- (knockout mice) Transgenic mice (simpler and can provide important information)-transgenic genes injected integrate randomly, integrated into pronuclei (so all of the genes, including germ line, will be altered)-transgenic reporter genes are important for understanding expression patterns-genes can be turned on at specific times in development and can be tissue specific (tissue-specific promoter) to deduce whether gene has activity in certain cell/tissue/organism...
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