Tissue-Specific KO of ECM Proteins - Methods in Molecular Biology: Extracellular Matrix Protocols - page 139

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Tissue-Specific KO of ECM Proteins
Emilio Hirsch, Mara Brancaccio, and Fiorella Altruda
1. Introduction
1.1. Gene Targeting
The analysis of phenotypes caused by null and mutant alleles is a very pow-
erful means to understand gene function in vivo. Historically, this experimen-
tal approach has been widely and successfully used in invertebrate models.
Now, thanks to the gene-targeting technology in ES cells, the genome of a
mammalian organism such as the mouse can be artificially modified by precise
alterations. The system exploits the ability of ES cells to be cultured and
manipulated in vitro without losing their totipotency (1 , 2) . Mutations in spe-
cific genes can be achieved by in vitro selection of ES cell clones in which the
locus of interest has been targeted by homologous recombination (3 , 4) . The
peculiar property of being totipotent allows ES cells, once injected in the cav-
ity of a blastocyst, to contribute to the formation of all cell types of a chimeric
embryo. Whenever a chimeric mouse possesses ES-derived germ cells, the
mutation can be propagated to its offspring. Heterozygous mice are then mated
to generate the homozygous offspring needed for phenotypic analysis.
1.2. Tissue-Specific Gene Targeting
Recent advances in gene-targeting technology in ES cells now allow induc-
ible mutations to be created in a tissue specific manner and at a precise devel-
opmental stage. Whereas the phenotype caused by germ-line mutations can be
biased by epigenetic adaptations, induction of gene alteration in differentiated
cells can result in clearer effects. Moreover, via this method, it is possible to
study the consequences of ablating genes essential for cell survival (5) , identi-
fying functions for distinct splice variants (6) , or tracking different gene func-
tions at two or more developmental stages (7) .