Filtration Of Transgenic Sheep Fibroblasts With SNanog-GFP

Since 2006,Yamanaka who pioneered an important research of reprogramming pluripotent somatic cells into pluripotent stem cells by means of pluripotency factors,people began to delve into all aspects of somatic cell reprogramming,one of which is about related pluripotent genes and their mechanism of action,such as Oct4,Sox2,Klf4,c-Myc,Nanog.Nanog gene has an important role in the maintenance of pluripotency and self-renewal of embryonic stem cell,and its significance has been a hot issue in the process of reprogramming somatic cell.The success of iPS from various species including human being was established,compared to which the research progress of sheep iPS is lagging behind,and rarely involved the use of sheep endogenous pluripotency gene.We cloned the core sequence of sheep Nanog promoter and connected it with pAcGFP1-N1 vector to build a eukaryotic expression vector in which the core sequence of sheep Nanog promoter could drivn the expression of reporter gene GFP.We used this vector to transfect sheep embryos fibroblasts,and screened it with G418 to obtain sheep Nanog-GFP transgenic cell lines.This study will further prove the role of Nanog in sheep somatic cell reprogramming and lay the foundation of sheep somatic cell reprogramming technology.The results of this study and the conclusions are as follows:1.Refer to sheep Nanog gene promoter sequence sented kindly by Professor Wang Wen from Kunming Institute of Zoology.CAS,we designed specific primers to colone the core sequence of sheep Nanog promoter from sheep genomic DNA.The length of the core sequence is 1836bp.2.We wiped off the CMV viral promoter of pAcGFP1-N1 vector,and link the remainder to sheep Nanog promoter sequence to construct SNanog-pAcGFP1-N1 eukaryotic expression vector.3.Expression activity of sheep Nanog gene promoter:The eukaryotic expression vector S-Nanog-pAcGFP1-N1 was transferred into sheep skin fibroblasts,mouse fibroblasts,mouse ES cells and mouse embryos to confirm that sheep Nanog gene 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-Nanog-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-Nanog-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,we have successfully screened transfer SNanog-GFP sheep embryo fibroblast cell lines.