Construction Of Eukaryotic Expression Vector With Bovine Gene Oct4/Sox2/c-Myc/Klf4 And Transfecting Skin Fibroblasts

Induced pluripotent stem (iPS) cells, which have the ability of self-renewal and potential to differentiate into a spectrum of adult cell types, can be generated from various somatic cells by the ectopic expression of transcription factors.Fibroblasts are spindle or flat stellate, with protrusions, which have more vigorous functional activity. But fibroblasts have a limited number of divisions, during the operation of transgenic experiments. This "short" the life of the body have a certain impact on producing transgenic animals through nuclear transfer, which limit their practical applications in research. In this study, bovine gene Oct4, Sox2, c-Myc, Klf4 were cloned from fetal bovine tissue and embryos. And the genes were cloned into the eukaryotic expression vector. The study provides the basis for the next research to obtain stem-like cells from fibroblasts by the use of iPS cell technology, which have self-renewal capacity of stem cell in a certain culture conditions, and ensure that gene targeting, screening and some preparatory work for the production of transgenic animals can be completed successful. Results are as follows:1. The open reading frame sequence of bovine gene Oct4/Sox2/c-Myc/Klf4 were cloned successfully with RT-PCR method from bovine fetal genital ridge and the blastocyst.The open reading frame sequence of target gene were cloned into pMD19-T vector by using gene recombination technology. E.coli (DH5α) were transformed with the recombinant plasmids, and then the positive clones were send for sequencing.2. The right open reading frame sequence of bovine gene were cloned into PIRES2-AcGFP1-Nuc vector by using gene recombination technology. 293FT cells were transfected with Recombinant plasmid. The expression of recombinant eukaryotic expression vectors were identified by Western blotting.3. Recombination plasmid transfected bovine fetal fibroblasts using FuGENE HD Transfection Reagent. The result show that the expression vector can express target gene effectively. The bovine fetal fibroblast were cultured by selection medium with G418. The result show that the selected concentration of G418 Bovine fetal fibroblasts was 700μg / mL, and the maintaining concentration was 350μg/mL.