Study Of Establishment Of Sheep Induced Pluripotent Stem Cells Via PiggyBac Transposition

Induced pluripotent stem cells(iPSCs)have the characteristics of self-renewal and pluripotent differentiation potential,and are a type of pluripotent stem cells.IPSCs are ideal cell models for biomedical and regenerative medicine research,and can be used to develop transgenic animals or protect excellent genetic resources in agriculture.Seven exogenous transcription factors(bovine OCT4,SOX2,KLF4,c Myc,porcine NANOG,Lin-28,SV40 Large T)were transferred into black bone sheep fibroblasts for Reprogram ming through piggyBac+TET-on transposition subsystem.The aim is to obtain a dark bone sheep iPSCs cell line with strong developmental potential,providing a theoretical basis for further research on the establishment of sheep pluripotent embryonic stem cells.The test results are as follows:The biological characteristics of fibroblasts derived from the skin of male and female black bone sheep were analyzed.The cell adhesion rate of low passages can reach95%,and the survival rate before and after cryopreservation can reach over 36%.The growth curve follows the normal cell growth pattern(S-type),with normal chromosome number and morphology(n=54,38/40)and no mycoplasma infection.It can be used as the starting cell line for inducing black bone sheep iPSCs.Black-bone sheep iPSCs induced by the piggyBac system can be continuously and stably subcultured on STO feeder layer cells for more than 26 generations.The AP staining part is positive,and the karyotype is normal(n=54,36/40).They express OCT4,SOX2,SALL4,NANOG,KLF4,and KLF17 pluripotent genes.In vitro culture,they can form embryoid bodies with the ability to differentiate into the inner,middle,and outer embryonic layers.Conclusion: By using the piggyBac+TET-on transposon subsystem,seven exogenous transcription factors(bovine OCT4,SOX2,KLF4,c Myc,porcine NANOG,Lin-28,SV40 Large T)can be transferred into black bone sheep fibroblasts to obtain sheep like iPSCs.