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) TRANSGENESIS: PRONUCLEAR DNA INJECTION
insert 300 copies of linear DNA (10-100kb) into ONE-CELL embryos
rarely will insertion alter other genes, but its possible
disadvantages: random insertion sites
any number might insert
proper regulation is difficult
advantages: faster than ES (no cell culture, no selection, no characterization)
preferred when SITE of integration is not crucial to understanding
PROCEDURE
1. mating of superovulating female mice and harvest of zygotes
2. use 400X microscope to microinject linear tgn DNA into pronucleus
3. transfer embryos to PSEUDOPREGNANT female (mated with a vasectomized
male mouse)
4. obtain tissue samples after pups are born and analyze with PCR
5. mate tgns to controls to establish mouse lines
mosaicism can occur if recombination takes place after one-cell stage
EXPERIMENTAL APPLICATIONS
1. analyze use of regulatory sequences (attach markers to promoters)
2. analyze effect of differential gene expression (muscle promoter for gene
normally expressed in fat)
3. DOMINANT-NEGATIVE tests of gene function (if dimers are required, a
mutated version of the gene will associate to prevent normal function)
5. models of dominantly inherited diseases (one copy can cause disease) CHIMERIC MODELS (2 different zygotes to make 1 embryo)
1) AGGREGATION chimeras – use acid to remove ZP from embryos at MORULA
stage and allow them t
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