Establishment And Characterization Of Stella-Cre Transgenic Mouse Line

Similarities in both the anatomy and physiology between the mouse and human make the mouse the most popular model organism used to study basic biological processes, development, immunology and behaviour. Mouse is ideally suited for genetic analysis because of its small size which allows mice to be housed in high density, its short generation time and prolificacy in breeding. Sequencing and analysis of the mouse and human genomes found that about 99% of mouse genes have at least one orthologue in the human genome. Thus the mouse represents a good model to study human gene function in vivo.Transgenic mice can be generated by pronuclear microinjection, viral vectors infection or gene targeting in mouse ES cells. Comparing gene targeting in ES cells, pronuclear microinjection or viral vectors infection both have some disadvantages such as transgene random integration, potential for mutating an endogenous gene. Thus, gene targeting in ES cells is the most safety and efficient way to generate transgenic mice.The Cre-loxP recombination system has been widely used to modify the mouse genome either in vitro or in vivo, including deleting, inserting or inversing specific genes or genome fragments and removing loxP-flanked selection markers. Cre-mediated deletion can be achieved by several methods such as:transient expression of Cre recombinase by a Cre-expression plasmid into mouse embryonic stem (ES) cell or the fertilized eggs; microinjection of the Cre mRNA into mouse oocytes or transduction of recombinant Cre protein into preimplantation mouse embryo; or mating with a Cre-expression transgenic mouse line.Depending on different porpurs, there are mainly three type of Cre transgenic mouse: tissue/cell-specific Cre transgenic line; inducible Cre transgenic line and general deleter Cre line.The development of recombineering technologies allows rapid construction of hundreds of conditional knockout alleles. Large genome-wide conditional knockout projects in the world such as European Conditional Mouse Mutagenesis (EUCOMM) and Knockout Mouse Project (KOMP) aim to make conditional knockout alleles for thousands of mouse genes. To study phenotypes of the null alleles, it is necessary to use a high efficient deleter line that expresses Cre transiently in the early embryos to avoid Cre toxicity from prolonged and high levels of Cre exposure. On the other hand, Cre expression in this deleter line should lead to efficient Cre-loxP recombination to convert the conditional alleles to null alleles.Here, we report a Cre-deletion transgenic mouse line (designated Stella-Cre) in strain 129S5/SvEvBrd (129S5), in which the Cre gene expression is under control of the endogenous mouse gene, Stella gene. Stella first highly expressed during oocytes maturation, and subsequently expressed in preimplantation embryos. The main results are:1) Using recombineering technology, we constructed an efficient Stella-Cre gene targeting vector;2) We generated a Stella-cre transgenic mouse by gene targeting and microinjection in 129S5/SvEvBrd (129S5) mouse line. And the gene targeting in ES cells were confirmed by using Long-Range PCR, data showed that the Cre expression cassette was targeted into 3’untranslate region (UTR);3) In our Stella-Cre transgenic mouse line, all the chimeras and their offspring appeared to exhibit normal viability and fertility, which means the Cre expression did not disrupt normal Stella gene function in these transgenic lines.4) The Cre expression was controlled by the expression of endogenous Stella gene, which means the Cre only short expressed in early embryos to avoid the Cre toxicity from prolonged and high levels of Cre exposure;5) In order to test Cre activity in vivo, we crossed the Stella-Cre mouse line with R26R reporter mouse line, in which the expression of lacZ will only be activated after Cre-mediated deletion of the floxed stuffer cassette in front of the lacZ coding sequence. PCR amplification, X-Gal staining, and FACS analysis were used to estimate the Cre-mediated deletion efficiency. Our data shown that, The Cre expression driven by endogenous Stella promoter showed efficient deletion activity in early embryos and germ line and the deletion efficiency is 100%.Thus, we generated a consistent Cre-deletion transgenic mouse line in 129S5. This transgenic mouse line could be used as a valuable tool to generate Cre-loxP mediated genomic alteration in vivo.