Filtration Of Transgenic Sheep Fibroblasts With S-Oct4-GFP

In recent years, the iPS technology has been developed rapidly, the major iPS-inducible factors have been cloned and iPS induction methods in animals (including humans) have been continually established. While the sheep iPS technology has lagged behind, and the iPS in sheep was mainly obtained by the use of heterologous (such as mouse) induction factors. In order to develop a sound sheep iPS technology, the sheep Oct4promoter was cloned with reference to bovine Oct4promoter primers and connected into pAcGFP1-N1vector to construct eukaryotic expression vector in which the expression of GFP was driven by Oct4. The expression vector was then linearized and transferred into sheep fibroblasts with the lipofection method so as to obtain positive cells, and lay the foundation for the establishment of a sound sheep iPS technology. The major results and conclusions obtained in this study are as follows:1. Sheep Oct4gene promoter cloning:with reference to the Oct4gene promoter sequences in GenBank, a1206bp fragment of Oct4gene promoter core sequence was amplified using the gradient PCR amplification which shares93%nucleotide identity with bovine Oct4promoter sequence.2. Construction of eukaryotic expression vector for sheep Oct4gene promoter:Eukaryotic expression vector S-Oct4-pAcGFP1-N1was constructed by connecting the sheep Oct4gene promoter and pAcGFP1-N1vector in which CaMV promoter had been deleted.3. Expression activity of sheep Oct4gene promoter:The eukaryotic expression vector S-Oct4-pAcGFP1-N1was transferred into sheep skin fibroblasts, mouse fibroblasts, mouse ES cells and mouse embryos to confirm that sheep Oct4gene promoter can be expressed in mouse embryos and mouse ES cells, but can’t in sheep skin fibroblasts and mouse fibroblasts.4. Transfection and screening of Sheep fibroblasts:Linearized eukaryotic expression vector S-Oct4-GFP was transferred into primary cultured sheep skin fibroblasts with the lipofection method and the positive transgenic cells were screened with G418.5. Identification of the positive cell lines:PCR method was used to identify positive transgenic cells, results showed that S-Oct4-GFP integrated into the transgenic cell genome successfully, cell morphology and cell growth curve of transgenic cells were the same as normally cultured cells. In addition, the transgenic cells still maintained normal cell morphology and proliferation rate after cryopreservation.In summary, sheep skin fibroblast cell line containing S-Oct4-GFP was obtained with the above methods.