Conditional gene targeting in mice has revolutionized the field of molecular biology and genetic engineering. In vivo applications of the Cre-lox system are highly efficient, specific, and versatile. The Cre-lox system is a powerful tool to manipulate and study gene function in vivo, and it relies on the Cre recombinase enzyme to induce site-specific recombination between two LoxP sites. This recombinase represents a critical molecular switch to activate or inactivate genes in selected cell types or developmental stages.
Cre recombinase is a site-specific recombinase that recognizes 34-bp DNA sequences called LoxP sites. Cre-mediated recombination occurs only between two LoxP sites that are oriented in the same direction and separated by an intervening DNA fragment. The net result of Cre-mediated recombination is excision, inversion, or translocation of the DNA fragment between the two LoxP sites. The efficiency and specificity of Cre-mediated recombination depend on several factors, including the Cre enzyme concentration, the LoxP site orientation and spacing, and the chromatin context of the targeted DNA sequence.
Cre-lox technology has been exploited to generate a wide range of Cre-expressing mice that can be used to drive Cre-mediated recombination in specific cell types or lineages. These Cre driver lines are often referred to as Cre lines and are widely available for researchers to use. Cre lines are generated by inserting a Cre-encoding DNA sequence into an endogenous gene locus or by using a transgenic approach to express the Cre enzyme under the control of a specific promoter. Cre lines have been developed for various cell types, including neurons, cardiomyocytes, hepatocytes, and immune cells.
One of the most commonly used applications of the Cre-lox system is gene floxing, which involves the insertion of LoxP sites into the genome of mice via homologous recombination. The LoxP sites flank the target gene or DNA fragment and allow for the selective deletion of the floxed gene upon Cre-mediated recombination. This approach is known as a conditional knockout, which allows the targeted inactivation of the gene in selected cell types or developmental stages. Conditional knockouts are a valuable tool for studying gene function in vivo, as they allow the researcher to assess the impact of gene deletion on specific cell types or tissues.
Another application of the Cre-lox system is conditional and inducible gene expression, which allows for the activation of a gene in response to external stimuli or at specific developmental stages. This technology is often used to control the expression of genes that are toxic, embryonic lethal, or essential for normal development. Inducible gene expression can be achieved using several approaches, including the tetracycline-inducible system, the estrogen receptor fusion protein system, and the Cre-ERT2 system. The Cre-ERT2 system uses a fusion protein of Cre and the mutated estrogen receptor ligand-binding domain, which allows for the control of Cre-mediated recombination by tamoxifen or its derivatives.
In summary, the Cre-lox system is a powerful tool for genetic engineering and gene function analysis in vivo. Cre recombinase is the key enzyme that mediates site-specific recombination between LoxP sites. Cre lines provide a means to drive Cre-mediated recombination in specific cell types or lineages. Gene floxing, conditional and inducible gene expression are some of the in vivo applications of the Cre-lox system that have been widely used in biomedical research. The Cre-lox system has also been used to create conditional and inducible gene knockout and knock-in mouse models, as well as cell-specific gene expression systems in rats. With such a versatile technology, researchers can now more effectively study gene function in vivo and unlock the mysteries of genetic disease and developmental biology.