Effects Of Suppressed The Expression Level Of Xist On The Expression Pattern And Developmental Efficiency Of Cloned Porcine Embryos

Somatic cell nuclear transfer(SCNT)has been used widely but circumscribed due to the abysmal cloned efficiency.It has been reported that both mutated XIST in mouse SCNT donor cells and suppressed the expression level of XIST using RNA interference(RNAi)in mouse pre-implantation embryos can improve cloned efficiency dramatically.In the previous study,a male porcine kidney cell colony containing a knocked-out XIST gene(XIST-KO)with an inserted EGFP gene at the exon 1 was generated by homologous recombination mediated by transcription activator-like effector nucleases(TALENs).In this study,two groups of SCNT embryo were generated using PK15-KO and wildtype PK15 cell line as donor cell separately,the expression level of X-linked gene was compared by transcriptome sequencing.Meanwhile,in this study,4 anti-XIST siRNA were transfected into female porcine kidney cell colony(PK21),one of the anti-XIST siRNA with the most interference efficiency was selected by the expression level of XISTat 24 h and 48 h after transfected.3 different kinds of RNAi including siRN A,siRNA with chemical modification and shRNA plasmid vector,were constructed and transfected into PK21 cell line,the most duration RNAi among was selected and was injected into cloned embryos at 2 cell stage.The results of this study were showed as followed:(1)shRN A plasmid vector has the longest interference effect compared to siRN A and siRNA with chemical modification.(2)Injected shRNA plasmid vector into cloned embryo at 2 cell stage can suppressed the expression level of XIST at blastocyst effectively compared to control group(P<0.05),the expression level of X-linked gene was up-regulated as well due to the interference of XIST.(3)Injected 10 p L 5ng/? L shRNA plasmid vector into cloned embryos at 2 cell stage cannot improve average number of cell in blastocyst and the blastocyst rate.(4)Using PK15 cell line with XIST mutated as donor cell,the expression level of X-linked gene was up-regulated(P<0.05).(5)Using PK15-KO cell line as donor cell finally decreased the developmental rates of cloned male porcine embryos.These results suggested that mutation of a XIST gene can only partially rescue abnormal XCI but cannot improve the developmental efficiency of cloned male pig embryos.The inability of XIST mutation to enhance the developmental potential of cloned pig embryos may be caused by incomplete resc ue of abnormal XCI,and/or by long-term drug selection of the nuclear donor cells which might have adverse effects on the developmental efficiency of mutated XISTpig embryos cloned from them.Injected shRNA plasmid vector into cloned embryo at 2 cell stage cannot improved average number of cell in blastocyst and the blastocyst rate may cause by the different pattern of XIST expression between different kind of animal.