| Somatic cell nuclear transfer can not only save endangered species but also has important application value in the fields of agriculture and medicine.However,the low efficiency of cloning has been restricting the development of cloning technology.Studies have shown that the Xist gene is abnormally highly expressed in cloned embryos,and the inactivation of the X chromosome is closely related to the Xist gene.Knockout of the Xist gene in cloned mouse embryos or inhibition of Xist gene expression by RNAi technique and RNAi combined with histone deacetylase inhibitor,mouse cloning efficiency was significantly improved.Previous studies in our laboratory found that injection of si RNA,into cloned pig embryos at the 1-cell stage showed a small improvement in the development efficiency of cloned pig embryos.It is still uncertain whether inhibition of porcine Xist gene can significantly improve the development efficiency of cloned pig embryos;inhibition of porcine Xist gene combined with HDACi treatment of cloned embryos can improve the efficiency of cloned pig embryo development and the superposition effect has not been reported.In this experiment,we compared the effects of two different RNA interference methods based on si RNA and based on sh RNA expression vector on Xist gene expression,X chromosome-linked gene expression and development efficiency of boar cloned embryos.We screened out the sh RNA interference method which is more beneficial to inhibit the expression of the Xist gene in boar cloned embryos,and then inhibited Xist gene expression by sh RNA or gene knockout in cloned pig embryos.At the same time,drug treatment of cloned embryos was used to inhibit histone deacetylation,and the effects of the two treatments on the development efficiency and gene expression pattern of cloned pig embryos were compared.The results are as follows:(1)Male porcine clone 2-cell stage injection of si RNA,could not inhibit Xist gene expression and save X chromosome inactivation in cloned embryos at blastocyst stage;(2)There was a significant difference in the average number of blastocyst cells of cloned pig embryos injected with sh RNA,in the male pig clone 2-cell stage,in which the blastocyst rate of sh RNA at the concentration of 5 ng/μl was close to the significant level,which could inhibit the expression of Xist gene and save the inactivation of the X chromosome in the cloned embryo blastocyst stage;(3)The best-combined treatment method for improving the development efficiency of cloned pig embryos in vitro is to knockout Xist gene and combine with LAQ824 to inhibit histone deacetylation.These results suggest that si RNA interference cannot inhibit Xist gene expression and rescue X chromosome inactivation in cloned embryos at the blastocyst stage,the interference mode of the injection of the anti-Xist sh RNA expression vector can inhibit the expression of the Xist gene and rescue the inactivation of the X chromosome in cloned embryos at the blastocyst stage.The treatment method based on the suppression of Xist gene expression combined with HDACi is beneficial to improve the in vitro development efficiency of pig cloned embryos. |
