Construction Of Expression Vector With Mouse Promoter UHS And Silk Dragline Protein Gene And Its Integration In Somatic Cells

In order to establish a new method to obtain the spider dragline silk from animal hair, mouse was chose as a animal model. By using spider dragline silk protein which was got from the preliminary work of the laboratory, mouse skin-specific promoter and recombinant plasmid named UHS-2S-3.1,CMV-2S-pIRES2-EGFP plasmid which is widely expressed in the body and the UHS-2S-pIRES2-EGFP plasmid which is not only expressed specifically in the skin but also can be easily detected by GFP reporter gene were constructed. Then mouse granular cells and mouse fetal skin fibroblasts were transfected by the recombinant plasmids respectively. G418 was used to filtrate the transfected cells and the anti-G418 cells were identified by PCR method to showed whether the vector constructed was integrated into cells genome. Results showed as follows:1.Construction of eukaryotic expression vector with silk dragline protein gene: polymerized spider dragline silk protein gene was linked to pIRES2-EGFP plasmid to get the expression vector CMV-2S-pIRES2-EGFP. Mouse skin-specific promoter gene was linked to the vector of CMV-2S-pIRES2-EGFP that wiped off CMV promoter after amplification in order to obtain the eukaryotic expression vectors with dragline silk protein UHS-2S-pIRES2-EGFP.2. Transfection and Filtration of mouse granular cells:granular cells from the young mouses were cultured in vitro to get the optimal cell line; After transfected linearization vector UHS-2S-3.1, the granular cells were filtrated by G418 and anti-G418 cells were identified by PCR method, the result showed that the vector constructed was integrated into granular cells genome.3. Transfection and Filtration of mouse fibroblasts:mouse fibroblast cell line were established by conventional methods; mouse skin fibroblasts were transfected with linearization vector CMV-2S-pIRES2-EGFP and UHS-2S-pIRES2-EGFP,then G418 was used to filtrate them. anti-G418 cells were identified by PCR and PCR identification of the positive cells indicated that objective gene integrated into genomic.These results of using original nuclear microinjection method or somatic cell nuclear transfer technology to get transgenic mouse which hair expressed spider dragline silk protein gene are conducive to establish a new method to get spider dragline silk protein gene from sheep hair. in the further research.