Study On Cloned Transgenic Rabbits

The efficiency of somatic nuclear transfer was significantly influenced by in vitro culture time and passages of donor cells. We carried on nuclear transfer with 5-10 low passage and 20-25 high passage of rabbit fibroblasts,, and found that the efficiency of donor nuclei are different between two experimental groups. Through comparing the rates of fusion, cleavage, blastocyst development and in vivo development of reconstructed embryos, we found that low passage of donor cell was more effective than high one, but normal cloned offspring could be prouduced from high passage of cells. This study confirms the influence of different passage cells of in vitro culture on the efficiency of rabbit nuclear transfer, and provides experimental basis and time window for gene targeting in rabbit.Somatic cell nuclear transfer has become the common techniques of producing transgenic animal, but the genetic modification of donor cells is an important factor which restricts the efficiency of transgenic animal production. This experiment was carried on as follows, to transfect the fibroblasts first with Metafectene Easy, then dilute them according to 1:40 and 1:80 densities, respectively. Selective agent G418 was not added until the confluency of fibroblasts reached 30%-40%, and eventually 41 colonies were selected, among them 12 colonies contain GFP. GFP colonies could be obtained from both densities,but the efficiency of positive expression from low density of cells is a little bit higher, therefore the density of 1:80 was more appropriate to screen transfeced cell. The results could provide a reference for improving transfection efficiency of cells, and production of transgenic animals.Cryopreservation of embryos was a very useful and indispensable tool in animal breeding preservation and distribution of laboratory animals. Vitrification was a kind of quickly freezing method independent of specific machines. In the present experiment, GFP transgenic and non-GFP morulaes selected under the fluorescence microscopy were vitrified. After thawing, 47%of the non-GFP embryos survived, and could develop to blastocyst stage in vitro. When the thawed GFP transgenic morulaes were transferred into recipient rabbits,21%(10/47) of them could develop in full-term. Four transgenic rabbits from vitrified embryos grow up to 4 months old. The results suggested that Vitrification could be used to preserve transgenic rabbit strains and improved the efficiency of embryos stocking by filtering the transgenic embryos.