Cre-Lox recombination

Cre-Lox recombination is a site-specific recombinase technology, used to carry out deletions, insertions, translocations and inversions at specific sites in the DNA of cells. It allows the DNA modification to be targeted to a specific cell type or be triggered by a specific external stimulus. It is implemented both in eukaryotic and prokaryotic systems. The Cre-lox recombination system has been particularly useful to help neuroscientists to study the brain in which complex cell types and neural circuits come together to generate cognition and behaviors. NIH Blueprint for Neuroscience Research has created several hundreds of Cre driver mouse lines which are currently used by the worldwide neuroscience community. Cre-Lox recombination is a site-specific recombinase technology, used to carry out deletions, insertions, translocations and inversions at specific sites in the DNA of cells. It allows the DNA modification to be targeted to a specific cell type or be triggered by a specific external stimulus. It is implemented both in eukaryotic and prokaryotic systems. The Cre-lox recombination system has been particularly useful to help neuroscientists to study the brain in which complex cell types and neural circuits come together to generate cognition and behaviors. NIH Blueprint for Neuroscience Research has created several hundreds of Cre driver mouse lines which are currently used by the worldwide neuroscience community. The system consists of a single enzyme, Cre recombinase, that recombines a pair of short target sequences called the Lox sequences. This system can be implemented without inserting any extra supporting proteins or sequences. The Cre enzyme and the original Lox site called the LoxP sequence are derived from bacteriophage P1. Placing Lox sequences appropriately allows genes to be activated, repressed, or exchanged for other genes. At a DNA level many types of manipulations can be carried out. The activity of the Cre enzyme can be controlled so that it is expressed in a particular cell type or triggered by an external stimulus like a chemical signal or a heat shock. These targeted DNA changes are useful in cell lineage tracing and when mutants are lethal if expressed globally. The Cre-Lox system is very similar in action and in usage to the FLP-FRT recombination system. Cre-Lox recombination is a special type of site-specific recombination developed by Dr. Brian Sauer that operated in both mitotic and non-mitotic cells, and was initially used in activating gene expression in mammalian cell lines (DuPont). Subsequently, researchers in the laboratory of Dr. Jamey Marth demonstrated that Cre-Lox recombination could be used to delete loxP-flanked chromosomal DNA sequences at high efficiency in specific developing T-cells of transgenic animals, with the authors proposing that this approach could be used to define endogenous gene function in specific cell types, indelibly mark progenitors in cell fate determination studies, induce specific chromosomal rearrangements for biological and disease modeling, and determine the roles of early genetic lesions in disease (and phenotype) maintenance. Shortly thereafter, researchers in the laboratory of Prof. Klaus Rajewsky reported the production of pluripotent embryonic stem cells bearing a targeted loxP-flanked (floxed) DNA polymerase gene. Combining these advances in collaboration, the laboratories of Drs. Marth and Rajewsky reported in 1994 that Cre-lox recombination could be used for conditional gene targeting. They observed ≈50% of the DNA polymerase beta gene was deleted in T cells based on DNA blotting. It was unclear whether only one allele in each T-cell or 50% of T cells had 100% deletion in both alleles. Researchers have since reported more efficient Cre-Lox conditional gene mutagenesis in the developing T cells by the Marth laboratory in 1995. Incomplete deletion by Cre recombinase is not uncommon in cells when two copies of floxed sequences exist, and allows the formation and study of chimeric tissues. All cell types tested in mice have been shown to undergo transgenic Cre recombination. Independently, Joe Z. Tsien has pioneered the use of Cre-loxP system for cell type- and region-specific gene manipulation in the adult brain where hundreds of distinct neuron types may exist and nearly all neurons in the adult brain are known to be post-mitotic. Tsien and his colleagues demonstrated Cre-mediated recombination can occur in the post-mitotic pyramidal neurons in the adult mouse forebrain. These developments have led to a widespread use of conditional mutagenesis in biomedical research, spanning many disciplines in which it becomes a powerful platform for determining gene function in specific cell types and at specific developmental times. In particular, the clear demonstration of its usefulness in precisely defining the complex relationship between specific cells/circuits and behaviors for brain research, has promoted the NIH to initiate the NIH Blueprint for Neuroscience Research Cre-driver mouse projects in early 2000. To date, NIH Blueprint for Neuroscience Research Cre projects have created several hundreds of Cre driver mouse lines which are currently used by the worldwide neuroscience community. Cre-Lox recombination involves the targeting of a specific sequence of DNA and splicing it with the help of an enzyme called Cre recombinase. Cre-Lox recombination is commonly used to circumvent embryonic lethality caused by systemic inactivation of many genes. In addition, Cre–Lox recombination provides the best experimental control that presently exists in transgenic animal modeling to link genotypes (alterations in genomic DNA) to phenotypes (the biological outcomes).