5-350 Lecture Notes - Blastocyst, Thymidine, Hypoxanthine-Guanine Phosphoribosyltransferase
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Published on 28 Jan 2013
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EMBRYONIC MANIPULATIONS
knockouts and transgenics
ANALYSIS OF GENE EXPRESSION
IN-SITU HYBRIDIZATION (RNA)
cross link proteins and remove lipids to allow access
use radioactivity, fluorescence or colorimetric assays
can be used to figure out TIMING of gene expression
IMMUNOHJISTOCHEMISTRY (PROTEIN)
location and timing of protein expression
MARKER/REPORTER GENES
product must be low BACKGROUND (specific) and low IMPACT (non-toxic)
lacZ (product is blue)
GFP – can be toxic, but also expressed as fusion-proteins
- requires that promoter for normal gene expression is known
- ligate reporter gene to the upstream controlling elements
- introduce marker gene/promoter to all cells (i.e. transgenesis)