| Research Abstract |
A transgenic mice line carrying a single copy of the transgene that is CAG promoter + lox71+bsr gene was established, and the homozygous male mice were produced through intercrossing. Fertilized eggs were collected from superovalated females crossed with the homozygous male mice, and injected with the Cre expression vector, pCAGGS-Cre, and the targeting vector that is lox66+NLSlacZ+MC1neo+pBluescript. When the targeting vector was inserted into the lox71 site in the mouse genome, the embryos were stained into blue with X-gal because the NLSLacZ gene was placed under the CAG promoter. The injected eggs were transferred into the oviducts of foster mothers, and the embryos were collected at 12.5 dpc, stained with X-gal. At the same time, the genomic DNAs were extracted from the placentas, and the integration pattern of the targeting vector was examined with PCR and Southern blotting. About 3500 eggs were microinjected under various conditions. Out of 486 embryos carrying the transgene, 5 embryos showed positive X-gal staining, demonstrating 1% of targeting efficiency. The DNA analyses of these X-gal positive embryos confirmed the targeted integration. However, the efficiency of 1% is not enough for effective production of transgenic animal. We are going to improve the system and raise the efficiency of targeting integration.
We have also produced transgenic mice lines carrying a lox71 site as promoter resources by gene trapping method. Until now, 17 mice lines have been established and analyzed the expression pattern of transgene.
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