Affects Of P53RNA Interference On The Development Of Early Bovine Somatic Cell Nuclear Transfer Embryos

Somatic cell nuclear transfer(SCNT), which is also known as somatic cloning, has greatpotential values in theoretical and practical applications, but the efficiency of SCNT is verylow and most cloned embryos cannot implant successfully or develop to term. What’s more,even live animals exhibit various developmental abnormalities. Increasing evidence showedthat low cloning efficiency was largely attributed to donor cell’s insufficient reprogrammingafter SCNT. In order to improve SCNT reprogramming, researchers have made great effort indonor cell selection and treatment, oocyte selection, improvement of SCNT technology, andmodifications of embryo culture procedures et al.. But up to date, the SCNT efficiencyremains low and there is plenty of room for improvement.p53, which is also known as protein53or tumor protein53, is a very importanttranscription factor encoded by the TP53gene. p53plays crucial role in a range of biologicalprocesses including apoptosis, cell cycle arrest/resumption, senescence, and mammalianreproduction et al.. Recent researches in iPSCs reprogramming found that depressing p53levels facilitated the reprogramming of somatic cells into induced pluripotent cells, indicatingthat p53protein posed a barrier to reprogramming. But in SCNT, however, the function of p53has not been reported previously. In order to investigate the function of p53in SCNTreprogramming, in this study we first constructed p53interfering vector, and obtained stablep53knocking-down cell strains which were used as SCNT donor cells subsequently. The maincontents and results of this study were as follows:1. Three interfering oligonucleotides targeted for p53mRNA and one negative controloligonucleotides were synthetized and inserted into pGPU6/GFP/Neo plasmid vector, oneshRNA plasmid vector TP53-300, which had the highest knocking down efficiency, werescreened by transient transfection;2. The interfering vector TP53-300was picked up and transfected into bovine fibroblastsfrom fetal skin, and four stably integrated clones were obtained by G418screening. Geneexpression of the four stable clones were detected by real-time PCR and results showed thatthe expression of MDM2, p21, Bax decreased and the expression of Bcl-2increased after p53knocking down. Flow cytometry analysis showed that apoptosis rate decreased, and cell cycleanalysis revealed that the ratio of G1phase increased while the ratio of S phase decreased after p53knocking down.3. In order to investigate the effect of p53knocking down on SCNT reprogramming,cells that were stably integrated with TP53-300plasmid vetor were used as donor cell, bovineoocytes matured in vitro as recipient cytoplasm, and then the cleavage rate, blastocystformation rate, blastocyst cell number and apoptosis rate of SCNT embryos were observed.Results showed that after p53interfering SCNT embryos’ cleavage rate increased, blastocystrate had no significant difference. Blastocyst cell number counting showed total cell numberof blastocysts increased after p53knocking down but the cell number of the inner cell massand trophectoderm had no significant difference, and apoptotic rate of blastocysts decreasedsignificantly.4. In order to further investigate the effects of donor cells’ p53interfering on SCNTreprogramming. The expression of development related genes NANOG, OCT4and SOX2were determined by real-time PCR and found the expression of NANOG decreased, theexpression of OCT4increased and the expression of SOX2did not differ significantly.Moreover, the CpG sites’ methylation status of three imprinting genes H19, IGF2R and XISTand three development related genes NANOG, OCT4and SOX2were determined by Bisulfitesequencing PCR(BS-PCR). BS-PCR analysis revealed that after p53knocking down, themethylation levels of H19and NANOG increased, the methylation levels of XIST and OCT4decreased, while the methylation levels of IGF2R and SOX2remained as previously.