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MBB331_Week9-Fall-2011

Blue staining lacz turned on by ie blue lacz enhancer

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Unformatted text preview: ressed & after XlacZ gal add’n, blue color indicates tissue-specific pattern of transcription gal controlled by enhancer… ie. blue staining = lacZ turned-on by ie blue lacZ enhancer (can also use anti-β -galactosidase antibodies to see enhancer -galactosidase expression pattern) expression ENHANCER TRAPS ENHANCER CNS & CNS neuronal lacZ lacZ enhancer traps in Drosophila embryos embryos Balakireva et al., (2000) J. Balakireva et ., Neuroscience 20, 3425. 20 • • • • enhancer trapping allows one to go after genes that may participate enhancer in the development of a particular tissue in for example, if you are interested in CNS development, go after for enhancers expressed in CNS enhancers clone out genes normally under control of these enhancers presence of reporter gene near enhancer provides “tag” reporter gene A number of approaches can be used to clone gene near enhancer: -make genomic library from transgenic animal and screen with probe from reporter gene or use inverse PCR: -genomic DNA from transgenic animal cut with r.e. that doesn’t cut in reporter gene, but will give some flanking genomic sequence -DNA fragments circularized by ligation and subjected to PCR using primers based on reporter gene that face opposite directions -inverse PCR amplifies flanking genomic sequences joined by r.e. site corresponding to original r.e. reporter gene (may be present with plasmid) -some Drosophila vectors used to insert transgenes also incorporate a complete bacterial plasmid into genome if plasmid present transform bacteria get “plasmid rescue” of flanking sequences -in this case, can begin as with inverse PCR technique, but after circularizing DNA, simply transform it into bacteria -this approach is called plasmid rescue and net result is isolation of a plasmid with flanking genomic sequences cloned into it ENHANCER TRAPS ENHANCER -as an example of enhancer trapping, it has been used to clone genes expressed at high levels in the CNS of developing mouse embryos DIRECTED GENE EXPRESSION IN Drosophila Drosophila Gene X = lacZ when checking enhancers • • • Drosophila Gal4p-Enhancer Trap: GAL4 gene inserted randomly into GAL4 genome (by P-element) and then crossed with flies carrying integrated UAS-lacZ promoter fusions integrated lacZ promoter Interesting cell specific lacZ expression patterns seen by lacZ Interesting lacZ lacZ staining identifies tissue-specific enhancers These enhancer GAL4 lines used to drive expression of UAS These regulated transgene (UAS-Gene X) regulated TRANSCRIPTION FACTORS: INDEPENDENT DNA BINDING & ACTIVATION DOMAINS DNA Lewin Genes...
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