BIOL 3010 Lecture Notes - Somatic Cell Nuclear Transfer, Zygosity, Gene Targeting
29 Jan 2013
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Embryonic manipulations: analysis of gene expression, rna expression: in situ hybridization. Fix sample, incubate with rna probe, detect probe: protein expression: immunohistochemistry. Fix sample, incubate with primary ab, wash, incubate with and detect secondary ab c) gene expression: reporter genes. Often use lacz gene (b -galactosidase) or green fluorescent protein (gfp) Fuse with gene of interest or create construct with sequences controlling expression of gene of interest. Transgenesis: pronuclear injection of dna: procedure: inject dna directly into nucleus, implant in pseudopregnant mouse, detect transgenic mice (e. g. southern blot) and breed, random insertion and requires many embryos, but is fast, applications. Deletion constructs of regulatory sequences to determine necessary sequences stages. Use alternative sequences controlling expression to get gene expressed in location where it is normally not. Test genes that whose function is eliminated by dominant-negative effect. Use reporter gene to mark a cell and allow identification at different.
