Abstract:

【Objective】This study aims to establish transgenic sheep fibroblast cell line and lay a foundation for transgenic sheep with expression spider dragline silk protein gene in hair follicle by nuclear transplantation.【Method】pcDNA3.1 and pGM-T-KAP6.1(hair follicle-specific promoter) were digested by BglⅡand Hind Ⅲ，and linked each other. The recombinant plasmid was linked with spider dragline silk protein gene and then linked with pIRES2-EGFP by a series of molecular methods. The eukaryotic expression vector pIRES2-EGFP-4S was constructed. Sheep fibroblasts were transfected with the plasmid by cationic liposome method and G418 was used to filtrate them. After identified, transgenic cell line with spider dragline silk protein gene was established. 【Result】The G418 positive cells were detected in vitro. The results showed that cellular morphology was similar with normal fibroblast, cell growth curve was “S” shape, population doubling time (PDT) shorten gradually along with culture time increase, plating efficiency was gradually increasing in 24h. These biological analyses showed the transgenic cells were similar with normal sheep fibroblast. Biological characters of the transgenic cells after freezing-thawing were similar with fresh sheep fibroblast. The PCR result showed that the vector constructed was integrated into sheep genome. 【Conclusion】The transgenic cells with polymerized spider dragline silk protein gene were filtrated, and thus laid a foundation for the transgenic sheep with expression spider dragline silk protein in hair follicle.