It is always necessary to carefully document the expression pattern of new Cre-driver mice. With method (a), insertion of Cre recombinase will likely disrupt expression of the gene, which is useful if one also wants to create a null allele with method (c) initiation of translation using the IRES will probably be less efficient than initiation of the primary mRNA. With all the methods described above, the insertion of foreign DNA (the bacteriophage Cre recombinase DNA) can influence expression of both the Cre recombinase and the gene into which it is inserted due to gene silencing (e.g. A cassette including Cre recombinase is typically inserted into the gene of interest either (a) just before the normal initiation codon, (b) after a 2A self-cleaving sequence that is inserted in place of the termination codon, or (c) after an internal ribosome entry site (IRES) sequence inserted just after the normal termination codon. A more reliable strategy involves gene targeting (by homologous recombination in embryonic stem cells or CRISPR/Cas9 methodology) such that a single copy of a Cre recombinase gene is expressed from an endogenous gene locus. These transgenic approaches can give cell-specific expression, but regulation of Cre recombinase expression may suffer from chromosomal effects due to the random site of integration and variable numbers of gene copies. A strategy that gives much more faithful Cre expression than this method involves inserting Cre recombinase into the gene of interest in a bacterial artificial chromosome (BAC) of about 200 kb and using that construct to generate so-called BAC transgenic mice, again with random integration. The first Cre-driver lines of mice had transgenes with relatively short regulatory regions fused to Cre recombinase, which integrated randomly into the mouse genome.
Controlling Cre expression using a cell-specific promotor allows selective inactivation, activation, or mutation of genes of interest. With the development of transgenic mice, where novel DNA sequences could be stably introduced into the mouse genome in a heritable manner, the Cre-lox system was first implemented in T-cells, and then in neuronal tissue. In 1987, the Cre-lox system was utilized for the first time in yeast, and subsequently in cultured mammalian cells. This site-specific recombination was initially discovered in bacteriophage P1 and the potential utility for cell-type-specific genetic manipulations was noted.
The lox songs list drivers#
By using cell-specific drivers for Cre, this recombination can be limited to generate conditional knock-out or knock-in animals. It initially creates a DNA loop and then either excises or inverts the looped segment depending on the orientation of the loxP sites. Cre recombinase allows cell-specific manipulation of gene expressionĬre recombinase (Cre) is a 343-amino-acid protein comprised of 4 subunits that recognizes pairs of specific 34 bp DNA sequences called loxP sites.
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