INDUCIBLE GENE DELETION BY CRE RECOMBINASE TRANSDUCTION

DESCRIPTION (provided by applicant): Genetic engineering in mice has allowed great advances in studies of in vivo biology. A number of technologies and tools have made this possible, from embryonic stem cells and the homologous recombination double-drug selection strategy, to more recent approaches such as the Cre/IoxP system. Cre is a DNA recombinase originally derived from the Escherichia coli (E. coli) P1 bacteriophage that recognizes specific 34 base-pair loxP sites of DNA recombination. The high specificity and efficiency of DNA recombination by Cre in a cofactor-independent manner attracted the attention of the research community as a means to modify the genome of higher organisms. The Cre/loxP system now has a growing number of uses such as conditional gene deletion, transgene activation or inactivation, and translocation between nonhomologous mouse chromosomes. A gene of interest can be deleted in whole animals, for example, by first engineering the gene to strategically incorporate loxP sites. Cre recombinase in the form of a transgene is then employed to mediate deletion of the gene via the loxP sites, perhaps in a cell-type specific or restricted manner. In this way, researchers can, for example, by-pass embryonic lethality that might occur with a conventional knockout mouse approach and/or dissect the in vivo role of a widely expressed gene in specific cell types. The investigators have now generated a novel reagent, TATCre. This protein represents a new approach to DNA recombination, in a temporally controlled fashion, by simply adding TATCre protein to cells. TATCre combines two previously described elements: 1) a peptide sequence identified in the HIV-1 TAT protein that is able to mediate receptor-independent transduction of heterologous proteins into cells (the 'transporter'); and 2) Cre recombinase (the 'cargo'). The investigators intend to develop this new research resource to the stage of being able to induce efficient and complete deletion of loxP-flanked targets in whole mice, by simply injecting mice with TATCre protein. Factors to be considered include the route, dose, and frequency of mouse injection with TATCre, as well as age and sex at the time of injection. The investigators also intend to continue the development of further Cre recombinase-transporter fusion proteins, including forms that employ polyarginine tracts as protein transporters in place of the TAT transporter. Thus, this proposal is intended to allow the development of Cre recombinase protein transduction technology. This approach offers the prospect of achieving induced gene deletion in whole animals and cell lines without the use of a Cre recombinase transgene, and, therefore, provides many more potential avenues for research.