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Outline of Lecture 04

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Biological Sciences
Asish Mukhopadhyay

Outline of Lecture 04 Embryonic Manipulations I. Analysis of Gene Expression A) RNA expression: in situ hybridization - Fix sample, incubate with RNA probe, detect probe B) 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 (β-galactosidase) or green fluorescent protein (GFP) - Fuse with gene of interest or create construct with sequences controlling expression of gene of interest II. Transgenesis: Pronuclear Injection of DNA A) Procedure: inject DNA directly into nucleus, implant in pseudopregnant mouse, detect transgenic mice (e.g. southern blot) and breed B) Random insertion and requires many embryos, but is fast C) Applications - Deletion constructs of regulatory sequences to determine necessary sequences - 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 stages - Mak
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