Biology Reference
In-Depth Information
The technique is based on the introduction of two or more short sequence
tags called loxP sites at specific positions in the genome. The P1 phage Cre
recombinase can recognize two loxP sites oriented in the same direction, cut
the intervening sequence, and rejoin the extremities (8) . Inducible gene target-
ing can thus be achieved by mating a mouse in which important sites in the
locus of interest have been flanked by loxP sites (a so-called floxed allele) with
a transgenic mouse that expresses the Cre enzyme in a restricted pattern (9-11) .
Similarly, the floxed allele can be silenced by infection with a virus that trans-
duces the Cre gene (12) . In this way, a variable percentage of cells ranging
from 10% to 100% (13) can be induced to undergo a controlled DNA rear-
rangement only when and where the recombinase is expressed.
1.3. Gene Targeting of ECM Proteins
Gene targeting has been widely used to study the function of ECM genes
(14) and these experimentally induced mutations greatly extended the knowl-
edge derived from the analysis of natural-occurring mutations (15) . Interest-
ingly, several of the knock-out mouse strains closely reproduce phenotypes of
human hereditary disorders (14 , 16-18) . In particular, mutations in ECM genes
expressed in bone and cartilage provide genetic models that clearly link muta-
tions to disease and greatly help to understand the molecular mechanisms
underlying congenital connective tissue alterations (14) .
Generation of mutant mouse strains has also resulted in the finding of new
and unexpected functions of ECM genes. For example, the analysis of
phenotypes of BM-40/SPARC and Thrombospondin 2-deficient mice drasti-
cally changed the view of their function (19 , 20) . These two proteins turned out
to be dispensable for morphogenesis but not for lens stability and lung homeo-
stasis, respectively.
On the other hand, genetic ablation of ECM components can result in an
unexpected lack of obvious phenotypes. Genes such as tenascin-C , although
showing a complex and highly regulated expression pattern during develop-
ment, are not essential for mouse development (21 , 22) . These observations
may suggest that some genes are of superfluous expression (23) , but also
that complex networks of compensation and functional redundancy are still to
be discovered.
2. Materials
2.1. Isolation of the Gene
1.
Cosmid library derived from 129/Sv mouse tissue.
2.
Kanamycin or ampicillin (cosmid dependent).
3.
LB: 10 g bactotryptone (Difco, Detroit, MI), 5 g bacto-yeast extract (Difco), 10 g
NaCl. Fill to 1 L with deionized water, adjust pH to 7.0, and autoclave. LB can be
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