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This lecture covers the different ways in which the mouse genome can be manipulated and then the consequences studied in the whole animal.
A predetermined endogenous gene can be altered in animals. This is effectively site-directed in vivo mutagenesis. Knockout or null mutations result in inactivation of the target gene.
These engineered animals are useful for studying gene function or loss of function in the whole animal. They can be used to correct a pathogenic mutation. Restoration of the normal phenotype could have therapeutic potential.
Learning objectives:
By the end of your revision, you should be able to explain how homologous recombination can be used to target site directed in vivo mutagenesis of specific gene loci. You should understand why this approach is taken and what information it can provide.
Background reading:
Human Molecular Genetics
T Strachan, AP Read
Chapter 21 pp 497-501.
Insights into cancer from transgenic mouse models
MacLeod KF, Jacks T
J. Pathol. (1999) 187:43-60.
Engineering the mouse genome by site-specific recombination
Metzger D, Feil R.
Current Opinion in Biotechnology (1999) 10:470-476.
Cre-mediated gene deletion in the mammary gland
Wagner K-U et al.,
Nucl. Acids Res. (1997) 25:4323-4330.
Main steps involved
Two main methods to target specific genes:
Insertional mutagenesis
Targets the locus of interest by a single reciprocal recombination
Methodology
Plasmid with:
A cloned gene or gene segment
A marker gene
Replacement mutagenesis
Some of the sequence in the chromosomal gene is replaced
This occurs as a result of double reciprocal recombination
Methodology
Plasmid with:
A cloned gene or gene segment
A marker gene within the homologous gene segment
Also
Double replacement gene targeting
Used to introduce more subtle mutations into the genome.
Methodology
Two plasmids:
First with two cloned introns that flank the exon to be mutated
Second with mutated exon flanked by the two introns
Introduction of DNA into ce
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