Establishment Of Induced Pluripotent Stem Cells Of Black-Bone Sheep

Pluripotent stem cells(PSCs)have unlimited self-renewal capacity and are able to differentiate into any tissue in an adult organism.Mammal pluripotent stem cells are generally classified into three types: embryonic stem cells(ESCs),induced pluripotent stem cells(iPSCs),and adult pluripotent stem cells.Inducing and establishing black-bone sheepiPSCs may provide a scientific basis for the optimization of sheepESCs in vitro.In domestic animals,iPSCs independent of exogenous genes have not been reported in cattle,goats and sheep,except pigs,so the induction of iPSCs in domestic animals needs to be further explored.We first established the black-bone sheepstarting cell line for iPSCs,used different transcription factor systems to reprogram somatic cells of black-bone sheepand try to obtain sheepiPSCs withstronger developmental potential or directed developmental potential.The cell biological characteristics of the obtained black-bone sheepiPSCs were studied from the aspects of cell morphology,karyotype,pluripotent gene expression,in vitro and in vivo differentiation ability and development potential.The result is as follows:1.In this experiment,two black-bone sheepfemale and male fibroblast cell lines were successfully isolated from the ear skin of 15 days old black-bone sheepby the tissue block culture method whichhave normal karyotype(2n=54),normal cell growthcurve(S type)and no mycoplasma contamination.It can be used as the starting cell line to induce i PSC in black-bone sheep;2.We designed and constructed three expression vectors PB-TRE-sLargeT(SV40 Large T),PB-TRE-hTERT and PB-TRE-sLhT(sLargeT and hTERT)based on the piggyBac+Teton transposition system.It could be integrated into the fibroblast genome of black-bone sheepby electro-transfection,and expressed the corresponding transgenic genes under the induction of doxycycline.3.X8(b OSMK+ pNhL+ sLhT)induction system,B9-LT(b OSMK+ pNhL+ hRL+sLargeT)induction system and B9-hT(b OSMK+ pNhL+ hRL+ sLhT)induction system can induce reprogramming of male black-bone sheepadult somatic cells,and SV40 Large T can increase the reprogramming efficiency.In this study,a total of 344 iPSCs cell lines of blackbone sheepwithcell clone morphology similar to mouse or human PSC were established,including 121 induced by X8 induction systems,134 induced by B9-LT induction systems,and 89 induced by 10 a induction systems;Two cell lines withthe highest degree of endogenous OCT4 activation,X8-4 and X8-6,were identified as siPSC-4 and siPSC-6.4.The black-bone sheepinduced pluripotent stem cell line siPSC-4 can be passaged stably for 49 generations,withnormal karyotype(2n=54),stable expression of endogenous OCT4,and expression of pluripotency genes OCT4,SOX2,NANOG and REX1 by immunofluorescence detection,have the ability to differentiate into three germ layers in vitro and formed teratoma withmesoderm and endoderm differentiation in vivo.In addition,we found that STO Feeder cells contribute to the continuous expression of SIPSC-4 endogenous gene OCT4,and the expression of SIPSC-4 endogenous OCT4 decreased under Feeder Free culture condition.Stem cell cultures 2i,t2 i,4i,5i of na(?)ve mouse and human na(?)vePSCs and expanded potential pluripotent stem cells(EPSCs)of mice could not maintain normal growthof siPSC-4 and ended in apoptosis.5.siPSC-4 has the potential to developin mouse and sheepearly embryos.The cells labeled td-tomato were detected in the ICM of mouse at 15%(3/20),26%(6/24)in the ICM of sheep.Importantly,we obtained a chimeric sheep(postnatal death)withdetectable expression of exogenous genes in the skin,cartilage,and kidneys of the sheep.In this study,we first used piggyBac+Tet-on transposable system to express extranetic transcription factors(bovine OCT4?SOX2?KLF4 and c MYC,porcine NANOG,human LIN28,SV40 Large T and human TERT)to induce the adult cells of male black-bone sheepto iPSCs.The iPSCs of black bone-sheepwere obtained,whichexpressed pluripotent genes and had the ability to differentiate into three dermal layers in vitro and in vivo and met the standard of pluripotent stem cells.This study provided an excellent experimental material for further study on the construction of ovine totipotent embryonic stem cells.