|Budget Amount *help
¥1,900,000 (Direct Cost : ¥1,900,000)
Fiscal Year 2004 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 2003 : ¥1,300,000 (Direct Cost : ¥1,300,000)
Genetic modification with Cre/loxP system in mice is widely used for the generation of tissue-specific knockouts and transgenics. However, tissue-specific genetic modification does not always rescue the mouse from unexpected embryonic death. In such cases, temporal regulation of Cre/loxP recombination is needed for Cre-expressing transgenic mice.
In our research, we used two types of tamoxifen-inducible Cre recombinase, MerCreMer and CreMer (gifted by Dr.Reth, Univ.of Cologne) for the generation of Cre transgenic mice. Five transgenic mouse lines expressing MerCreMer under the control of GAG promoter (gifted by Dr.Miyazaki, Osaka Univ.) were generated with pronuclear DNA microinjection to C57BL/6J fertilized eggs. Two lines, in which MerCreMer protein expression was showed by Western blot analysis, were crossed with ROSA26R mice (purchased from Jackson laboratory). However, X-gal staining analysis of MerCreMer/ROSA26R doubly transgenic mice did not show tamoxifen-dependent cre-mediated
excision of foxed ROSA26 gene. MerCreMer expression in the two lines was not enough to induce tamoxifen-dependent recombination.
Next, we planned to generate transgenic mouse lines expressing CreMer strongly and ubiquitously. We constructed the transgene harboring GAG promoter, "frted" GFP-neo fusion gene and CreMer gene. The neo gene was derived from mutated MC1-neo-pA cassette. The transgene was electroporated into E14.1 ES cells. The embryoid bodies derived from three G418-resistant ES clones expressed GFP strongly and ubiquitously. The transient expression of FLP recombinase in the three clones induced the excision of feted GFP-neo gene and subsequent CreMer expression. ES cells before FLP/FRT recombination were introduced into C57BL/6J blastocysts. One of those clones, No.32, was germline-competent and the transgenic mouse line, CM 32, was established. Many tissues such as skeletal muscle, heart, pancreas, skin and intestine, were GFP-positive in CM32 mice. We are planning to cross CM32 mice with FLP66 mice (Takeuchi T. et al.,2002). Less