Establishment Of PiPSC And Activation Of Pig Reprogramming Factors By CRISPR

Somatic cells can be reprogrammed into induced Pluripotent Stem Cells(i PSC)with the overexpression of key reprogramming factors.IPSC have self-renewal capacity and can differentiate into three germ layers in vivo and vitro.IPSC offer great potential for regenerative medicine.Pig are valuable animal models in pre-clinical research due to their anatomical and similarity to human-beings.Therefore,it is important to study Pi PSC.But Pi PSC have a problem of low efficiency.In this study,we aimed to improve the Pi PSC efficiency:(1)MEF(Mouse Embryonic Fibroblasts)cells can be induced into i PSC by i CD1 medium efficiently and quickly.We try to induce Pi PSC by i CD1 medium and compared efficiency with the previously reported MXV medium.(2)CRISPR/d Cas9(Clustered Regular Interspaced Short Palindromic Repeats/dead associated protein9)can activate gene expression.We used the SAM(Synergistic activation mediator)activation system select efficient sg RNA(Single-guide RNA)for target activating porcine Oct4,Sox2,Klf4 and c-Myc(OSKM)and mi R-302/367.Obtaining combine five sg RNAs tandem vector to simultaneous activation of porcine endogenous OSKM and mi R-302/367 transcription,preparing for reprogram PEF cells to obtain Pi PSC.The main findings are as follows:1.PEF cells can be reprogrammed into Pi PSC with i CD1 Medium.PEF cells were incubated with the lentiviral of GV358-OSKM and FUGW-mi R-302/367,and then PEF cells were reprogramed with i CD1 Medium.Pi PSC clones were generated and passage,and which was positively stained against alkaline phosphatase.2.Pi PSC clones reprogramming are more efficient and shorter time in i CD1 medium than in the MXV medium.Pi PSC clones need to be cultured in the i CD1 Medium for 10 day,and the Pi PSC clones need 16 day in MXV medium.The number of Pi PSC clones in i CD1 medium were three times that of MXV medium.3.PK-15-d Cas9-MS2 cells were selected to increased activation efficiency of the SAM system.PK-15 cells were used to incubate lentiviral of lenti d CAS-VP64?Blast,lenti MS2-P65-HSF1?Hygro for 2 days,then these cells were treated with blasticidin(10?g/m L)and hygromycin(500 ?g/m L)for 6 days,the PK-15 cell line was obtained with stable expression of lenti d CAS-VP64?Blast and lenti MS2-P65-HSF1?Hygro,named PK-15-d Cas9-MS2.4.Six or ten sg RNAs were designed to target porcine reprogramming factors OSKM and mi R-302/367 in their promoter region.Each sg RNA was ligated into lenti sg RNA(MS2)?zeo backbone vector.Seven Sg RNAs plasmids were constructed to target Oct4 and c-Myc genes,six sg RNAs plasmids were constructed to target Sox2 and Klf4 genes,and ten sg RNAs plasmids were constructed to target mi R-302/367.The luciferase reporter plasmids of OSKM and mi R-302/367 were generated by PCR amplification and ligation with p GL3-basic plasmid.5.Dual luciferase assay was used to detect the efficiency of different sg RNA in PK-15-d Cas9-MS2 cell.Targeting OSKM and mi R-302/367 sg RNAs were tested actively,these sg RNAs targeting in different gene region had different editing efficiencies.We selected two highly active sg RNAs.6.Gene promoter can significantly be activated by single sg RNA or simultaneous expression two sg RNAs.However,promoter activity was higher by two sg RNAs than single sg RNA.Further RT-q PCR detection showed that the expression levels of target genes and mi RNA were significantly increased when two sg RNAs were simultaneous expressed.7.To test whether the SAM activation can simultaneous activate promote activity of OSKM and mi R-302/367.We construct a tandem sg RNAs vector.The dual luciferase assay showed that the plasmids of tandem sg RNA could activate the transcriptional activities of OSKM and mi R-302/367 in PK-d Cas9-MS cells.In summary,Pi PSC clones reprogramming are more efficient and faster in i CD1 medium than in the MXV medium.ICD1 medium provides a new method for Pi PSC.We selected efficient sg RNA(Single-guide RNA)for activating porcine OSKM and mi R-302/367 by SAM activation system and obtained the plasmids of tandem sg RNA in this study.The strategy of tandem sg RNA provides a new idea for reprogramming PEF cells to obtain Pi PSC.