The Establishment Of Transgenic Sheep Fiber Cell Strains With Kap6.1-pcDNA3.1-2S

The spider silk possesses incredibly excellent property; meantime it acts as complex material with layered structure. Therefore, numerous researchers are paying attention to this stuff. Because of the highly territorial awareness and its aggressiveness, difficulties can be seen in artificially feeding spiders. This research used the keiatin associated protein gene Kap6.1as promoter; meanwhile connecting it with pcDNA3.1with neo resistance to construct the eukaryotic expression vector, then connect with the synthesized spider silk protein dimmers to constructing the eukaryotic expression vector which can start the spider silk protein, and transfected the skin fibroblasts in sheep. After screen the positive cell through G416, and enlarge the calculation, we have established the transgenic cell strain of sheep skin fibroblasts with spider silk protein dimmers, which lay the foundation of latter experiments on researching and producing transgenic sheep.The results and conclusions:1. Construction of eukaryotic expression vector:With CMV promoter be removed, pcDNA3.1was connected to the promoter Kap6.1digested by the same enzyme and then to spider silk protein dimer2S.The eukaryotic expressing vector Kap6.1-pcDNA3.1-2S was constructed.2. Transfection and screening of sheep skin fibroblasts:After linearizing Kap6.1-pcDNA3.1-2S, transfecting sheep skin fibroblasts with liposome method. Screening with the proper G418concentra tion to get the neo resistant cells.3. Detection of the positive transgenic cells:extracting the DNA of genetically modified positive cells, testing with PCR methods. The results showed that the linearized Kap6.1-pcDNA3.1-2S successfully intergrated into the DNA of the sheep skin fibroblasts; meantime the cells’ shape and growth curve are in accordance with the normal cells. The transgenic cells’growth status remain well after post-thaw.