Study On Establishment Of Black Bone Sheep Induced Pluripotent Stem Cells By 10 Transcription Factors

Induced pluripotent stem cells(iPSCs)refer to the ability to self renew and differentiate into other types of cells by introducing specific transcription factors into animal cells to make differentiated somatic cell reprogramming.Compared with embryonic stem cells(ESCs),somatic cell cells used to generate iPSCs come from a wide range of sources,avoiding ethical problems caused by embryo damage.Therefore,they have great application potential in drug research and development,disease treatment and animal breeding.However,the pluripotent regulatory mechanism of cells has not been fully elucidated,which hinders the acquisition and application of i PSCs in production practice.This study used the method of electric transfection to induce the generation of i PSCs from 15-day-old fetal ear fibroblasts of black bone sheep,and measured the biological characteristics of the obtained i PSCs.The aim was to explore the establishment and improvement of an efficient method for obtaining sheep i PSCs.The specific results are as follows:1.Successfully resuscitated female and male black bone sheep ear fibroblasts,with normal karyotypes(82% for female and 78% for male black bone sheep ear fibroblasts),negative Mycoplasma detection,and good growth status,showing a typical "S" shaped growth trend.2.Using the U203 program,PB TRE bOSKM(bovine OCT4,SOX2,cMYC,KLF4),PB TRE p Nh L(porcine NANOG and human LIN28),PB TRE h RL(human RARG,LRH1),PB TRE s Lh T(simian virus Large T and human TERT)expression vectors were electrotransformed and integrated into the genome of black bone sheep ear fibroblasts to obtain black bone sheep iPSCs.At the same time,the corresponding exogenous genes are expressed by i PSCs cultured in the cell culture medium added with of 1.0μg/m L Doxycycline.3.The black bone sheep iPSCs-15 established in this experiment can be stably cultured until 20 generations,with normal karyotype(karyotype normal rate: 74%),and stably expresses core pluripotent genes such as endogenous OCT4,SOX2,NANOG,SALL4,TFCP2L1,etc.Immunofluorescence shows positive OCT4,SOX2,and NANOG proteins.In vitro differentiation test showed that the i PSCs obtained had the ability to express three germ layer genes,and could differentiate into teratomas with mesoderm and ectoderm in vivo.4.The analysis of transcriptome data shows that TGF is present in the KEGG signal pathway with significantly enriched differential genes of i PSCs generated by the ear fibroblasts of black bone sheep-β Signal pathway,PI3K/AKT signal pathway,Wnt signal pathway,etc.These pathways may play an important role in cell proliferation and pluripotency maintenance...