Screening And Evaluation Of MiRNAs Promoting The Production Efficiency Of Sheep IPSCs And Directional Differentiation Research Of Female PGC-like Cells

Sheep is a very important domestic animal.It is a direction to improve or breed sheep by means of gene modification.At present,it is difficult to control exogenous genes and low efficiency in molecular breeding using transgenic technology.Gene targeting based on mouse embryonic stem cells(ESC)enables precise and efficient knock-in and knock-out.Due to the difficulty in isolating and obtaining sheep ESC,the use of induced pluripotent stem cells(i PSCs)to replace ESC for precise genetic engineering has become an urgent problem.At present,low induction efficiency of sheep i PSCs is the problem and it is difficult to obtain fully reprogrammed i PSCs.microRNAs are post-transcriptional regulators of gene expression,and ESC-specific miRNAs have been shown to promote somatic reprogramming.Therefore,screening miRNAs that can promote somatic cell reprogramming in sheep and exploring their reprogramming mechanisms are expected to establish a highly efficient system to obtain sheep i PSCs,which has application value for high-quality sheep breeding and also lays a foundation for the acquisition of sheep ESC.Germ cells are the only cell lineages that transmit genomic and epigenetic information to the next generation,but reconstructing germ cell development has always been a challenge for biological research in vitro.As mouse pluripotent stem cells can differentiate into primitive germ cell like cells(PGCLC)in vitro,and then develop into oocytes and could carry on germ line passage,which provided a theoretical basis and method for the production of PGC from sheep pluripotent stem cells in vitro.The study on the directional differentiation of sheep i PSCs into female PGCLC can provide a cell model for the development of sheep PGC and lay a foundation for the later differentiation of functional ovum,which has important application value for improving the use of high-quality gametes in sheep and accelerating the improvement speed of herds.Objective:(1)Screening microRNAs that can promote somatic cell reprogramming in sheep;(2)To preliminarily explore the mechanism in the process of somatic reprogramming using microRNA in sheep;(3)To establish a method for inducing differentiation into primordial germ-like cells using sheep i PSCs.Methods:(1)Sheep fetal kidney cells(SKC)of ?45 days of gestation were isolated and cultured by tissue block method and used as the starting cells for reprogramming.Fetal fibroblasts(MEFs)of CF-1 mouse were isolated by trypsin digestion and treated with mitomycin C as feeder cells to induce sheep i PSCs.(2)Combined with literature reports and database,comprehensive analysis of miRNAs that are highly expressed in mouse and human stem cells or can promote reprogramming.Clusters miR302/367,miR200b-200a-429 and miR200c-141 that could promote somatic cell reprogramming in sheep were screened and packaged into lentiviruses.Eight human transcription factors including OCT4,KLF4,SOX2,C-MYC,Nanog,LIN28,SV40 T and h TERT were packaged into lentivirus.SKC was infected with different lentiviruses for induction reprogramming.The induction efficiency was calculated by alkaline phosphatase(AP)staining to screen out the best miRNA that could promote somatic reprogramming in sheep.The obtained sheep i PSCs were identified from the aspects of AP staining,real-time quantitative PCR,cell immunofluorescence,karyotype analysis,methylation sequencing and embryoid differentiation.(3)Bioinformation platforms such as miRbase and Target Scan were used to predict the target genes of miR-200 c,then oar-miR-200 c mimics,inhibitor and negative control were synthesized in company.Primers were designed to clone 3' UTR regions of target gene binding sites and 3' UTR mutations,and dual luciferase reporter vectors for target genes ZEB1 and TWISTNB were constructed.(4)oar-miR-200 c mimics were transfected into cells,and the interaction with target genes was analyzed using luciferase reporter vector.Transcription level of target genes was detected by qRT-PCR.Western blot was used to detect the protein levels of target genes.To investigate the mechanism of oar-miR-200 c in the reprogramming process of sheep cells.(5)Primers were designed to amplify and clone the reproductive related genes of DAZL and BOULE in sheep.Lentiviral vectors PLVX-DAZL and PLVX-BOULE were constructed,and the lentivirus was packaged.(6)Sheep ovary primary cells were isolated and cultured by tissue block method.Primordial germ cells were differentiated with sheep i PSCs by exploring the culture system and conditions.The transcription level of sheep germ cell specific factors was detected by qRT-PCR,and the protein level of sheep germ cell specific factors was detected by Western blot.Results:(1)SKC was successfully isolated from sheep somatic cells and expressed keratin CK18.Mouse CF-1 MEF cells were successfully isolated and feeder cells were prepared,which could well support the growth of si PSC.(2)Three screened miRNA clusters were successfully packaged with miRNA lentiviruses.Eight transcription factors were successfully packaged into lentiviruses.After the infection of sheep SKC with lentivirus,miR200c-141 was selected to increase the reprogramming efficiency to 0.95% through comparative analysis of induction efficiency.The obtained sheep i PSCs were identified as positive.(3)Dual luciferase reporter vectors with target genes ZEB1 and TWISTNB of miR-200 c were successfully constructed;(4)Dual-luciferase reporting analysis showed that oar-miR-200 c targeted the 3'UTR of ZEB1 and TWISTNB;qRT-PCR and Western blot detection showed that the expression of ZEB1 and TWISTNB genes was significantly decreased by oar-miR-200 c,but increased the expression of E-cadherin.oar-miR-200 c enhances MET transformation by affecting TGF-? signaling pathway,thus improving somatic cell reprogramming efficiency in sheep.(5)Lentivirus vectors LVX-DAZL-F2A-m Cherry IRE-neo and PLVX-Boule-F2 Am Cherry were successfully constructed,and lentivirus was packaged;(6)Sheep i PSCs with stable transfection of DAZL and BOULE genes were successfully established.Adding into Activin A,b FGF,BMP4,LIF,EGF and other factors as well as the supernatant of ovarian cell in culture medium,Sheep i PSCs differentiated into PGC-like cells.PGCLC could express reproductive related marker proteins PRDM1,PRDM14 and VASA,and the transcription level of DAZL,BOULE,PRDM14 and VASA showed high on day 8.Conclusion:(1)miR-200c-141 was successfully screened to promote the reprogramming of sheep somatic cells to i PSCs;(2)oar-miR-200 c targeted ZEB1 and TWISTNB genes,significantly decreased the expression level of target genes and increased the expression of E-cadherin;oar-miR-200 c enhances MET transformation by affecting TGF-? signaling pathway,thus improving somatic cell reprogramming efficiency in sheep.(3)Sheep i PSCs were infected with lentivirus of germline related genes DAZL and BOULE,and could be differentiated into primordial-germ like cells by adding activin A,b FGF,BMP4,and LIF etc.