Filtration Of Transgenic Sheep Skin Fibroblasts With Polymerized Spider Dragline Silk Protein Gene

Spider dragline silk is a one of the most elastic and tenacious proteinaceous fibers with remarkable mechanical properties which make it attractive for technical applications. Unfortunately, because the spiders can not live together, the material cannot be obtained in large quantities. So the vector of the wool keratin promoter which was linked with artificial synthesized spider dragline silk protein was transfected to sheep skin fibroblasts. G418 was used to filtrate the transfected cells and the anti-G418 cells with neo gene gained which were the transgenic cells. Therefore the cells could be used in the study of establishing transgenic sheep fibroblast cell line and the transgenic sheep by nuclear transplantation. Main results and conclusion were showed as follows:1. Clone of sheep keratin promoters: according to the sequences in the GenBank, keratin promoter(K2.10),high glycine/tyrosine protein gene promoters(KAP6.1) and high sulphur keratin gene promoters(B2A/B2C) were cloned by PCR technology respectively.2. Expression activity of sheep keratin gene promoter: before-mentioned promoters were respectively linked with GFP report gene to get expression vectors. Sheep fibroblasts were transfected with these vectors. The results showed that K2.10, KAP6.1 and B2C promoters had expression activity in sheep fibroblast, and the B2A promoter hadn't. The K2.10 promoter was selected to construct eukaryotic expression vector.3. Construction of eukaryotic expression vector with sheep keratin promoter: K2.10 was linked with pcDNA3.1 that wiped off CMV promoter, and then eukaryotic expression vectors with sheep keratin promoter were obtained.4. Construction of eukaryotic expression vector with dragline silk protein gene: polymerized spider dragline silk protein gene was linked to expression vector with sheep keratin promoter. A pair of particular primers was to obtain the sequences of silk protein and keratin promoter gene. The sequences were linked with the vector of pIRES2-EGFP that wiped off CMV promoter, and then eukaryotic expression vectors with dragline silk protein were obtained. The vector was linearized with restriction enzyme.5. Transfection and Filtration of sheep fibroblasts: sheep skin fibroblasts isolated from fresh were transfected with linearization vector by cationic liposome method and used G418 to filtrate them. Anti-G418 cells were gained.6. Identification of anti-G418 with neo gene: anti-G418 cells were identified by PCR method, the result showed that the vector constructed was integrated into sheep genome. The fibroblast cell growth curve, the cell planting survival efficiency and the cell population doubling time of anti-G418 cells were analyzed. At the same time another fibroblast cell growth curve was tested after the cells were frozen and thawed. And all the results tallied with the normal law, suggesting that the transfection and the freezing-thawing didn't have any effects on cells.In conclusion, transgenic cells with polymerized spider dragline silk protein gene were filtrated.