5-350 Lecture Notes - Zygosity, Somatic Cell Nuclear Transfer, Gene Targeting

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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
- Make models of dominant genetic diseases
III. Transgenesis: Chimeras
A) There are two ways to make chimeras
1) Aggregation: let two morulas fuse, implant in pseudopregnant mouse
2) Blastocyst Injection: inject ES cells into blastocyst cavity, implant
B) Important aspects of these chimeras
- Extensive intermingling occurs, with unpredictable partitioning in each
organ
- Normal mice result
IV. Embryonic Stem Cell Technology
A) ES cells are derived from ICM and cultured with appropriate factors (e.g. LIF)
B) ES cells are pluripotent in all aspects
C) Breeding germ-line competent chimeras involves different coat colors and
DNA analysis
D) Gene targeting (by G418 positive selection and gancyclovir negative selection)
is powerful way to make heterozygous knockouts or knockins
E) Role of embryonic lethal genes can determined by observing time of lethality
F) ES -/- cells (three ways to make from +/- cells) are experimentally useful
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