Effects Of Inhibition Of H3K9me3 Histone Methylation On In Vitro Developmental Ability Of Porcine Cloned Embryos

The pig cloning technique is also called somatic cell nuclear transfer(SCNT). It has valuable applications in pigs' breeding, reproduction, genetic resource preservation, human biomedicine, and life science. However, current success rate of pig SCNT is about 1%(number of born cloned pigs/number of transferred cloned embryos), and such a low efficiency seriously limits the improvement and applications of the SCNT technique.The main cause leading to the low cloning efficiency is the reprogramming abnormalities of the cloned embryos. Therefore, transcriptional regulation of the developmentally important genes may improve the pigs cloned embryos developmental abilities.This study aims to remove the histone methylation H3K9me3 ofthecloned porcine embryos by donor cells followed by SCNT transfection of SUV39H1/H2(histone methylases of responsible for establishing the H3K9me3) siRNA, or pigs cloned embryos microinjection of KDM4A/B/D(histone demethylases of specificlly removing H3K9me3) mRNA and explore effects of the two methods on the rate of the cloned embryos in vitro developing to the expanded blastocyststage.In this study, we constructed three expression vectors, including the homo sapiens vector KDM4A-pcDNA3.1(+),mice vectors KDM4B/D-pcDNA3.1(+), and acquired the mRNA of the KDM4A/B/D genes by in vitro transcription. Then,we microinjected the KDM4A/B/D mRNA to the 1-cell stage porcine cloned embryos cytoplasm. Meanwhile, we synthesized and selected the optimal SUV39H1/H2 siRNAs, then co-transfected the siRNAs to the donor cells followed by SCNT successive transits. We observed and counted the rate of the cloned embryos of in vitro culture to the expanded blastocyst stage by the two ways and randomly selected several embryos in 24 h for testing the expression level of the histone methylation H3K9me3 by the immunofluorescence experimental technique. The experimental results showed that:(1) The blastocyst rate of the porcine embryos can be partly improved by the way of microinjection of KDM4A/B/D mRNA into the 1-cell stage cloned embryos, but the difference is not significant; The blastocyst cell numbers were significantly increased in the KDM4 B mRNA injection groups.(2) the porcine cloned embryos' blastocyst rateand the blastocyst cell numbers by in vitro culture can be partly improved by the way of donor cells followed by SCNT co-transfection of SUV39H1/H2 siRNAs, but the difference is not significant;(3) It cannot efficiently remove the H3K9me3 mark by the way of the mRNA injection and siRNA interference.