Biology Reference
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14
Homologous Gene Targeting
to Study ECM Assembly
Francesco Ramirez, Friedrich Laub, and Hideaki Sumiyoshi
1. Introduction
Genetically engineered mutant strains of mice have become invaluable tools
to decipher complex biological processes, such as ECM assembly, and to dis-
sect phenotypic overlaps, like the genodermatoses. Homologous gene target-
ing in embryonic stem (ES) cells is increasingly favored over the transgenic
approach because it allows the study of mutations in homozygosity and with-
out altering the remainder of the genome
(1)
. Homologous gene targeting has
been successfully employed to abrogate the expression or alter the structure of
ECM-coding genes
(2
,
3)
. In both cases, the experimental protocol consists of
the following three steps: a. construction of the targeting vector; b. homolo-
gous recombination in ES cells; and c. generation of mutant mouse strains.
The design of the targeting vector requires extensive knowledge of the dis-
tribution of restriction sites, the organization of the exons and introns, and the
location of repetitive sequences within the genomic region in question. The
ideal targeting vector consists of
5 kb of genomic sequences (homology arms)
flanking the area to be targeted and where the promoter/gene-cassette for a
positive selectable marker (neomycin/G418) has been placed. The neo-cassette
contains diagnostic restriction endonuclease sites to verify the recombination
by Southern blot analysis of ES-cell DNA. Traditionally, a promoter/gene-cas-
sette for a negative selectable marker (HSV thymidine kinase/Gancyclovir) is
placed immediately outside of the homology arm(s) to select against random
insertion of the targeting construct into the genome
(1)
. Depending on the
design, the neo-cassette can put the endogenous gene out-of-frame (insertion
targeting; null mutation) or substitute one or more exons without altering its
open-reading-frame (deletion targeting; structural mutation)
(1-3)
.
≥
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