| The successful production of cloning mammals needed to reprogramme the nuclei of somatic cell efficiently. Reprogamming was refered to erase the epigenetic marks of somatic cell, convert it to earlier and undifferentiated state. H3K9me3-dependent heterochromatin was a barrier of reprogramming of cell identity. Suppressor of variegation3-9homolog1/2, SUV39H1/2 genes coded specifical H3K9 histonmethyltranferases, promoted the compassion of chromatin and the formation of pericentic heterochromatin, and regulated gene transcription properly. In this study, the expression of SUV39H1/H2 genes was downregulated in Debao pig ear fibroblasts by infecting high titer lentivirus containing their shRNAs. Donor cells that SUV39H1/H2 genes knockdown were selected and injected into the cytoplasm of an enucleated egg. The objective was to determine the effect of SUV39H1/H2 genes downregulation on development potentiality of pig SCNT preimplantation embryos. The results were looking forward to improving the efficiency of cloned animals.Construction of SUV39H1/H2 expression vectors and verification of shRNAs inhibition efficiency1. In order to obtain shRNAs with significant inhibition efficiency, exon 2 sequence of Debao pig SUV39H1 gene and ORF sequence of SUV39H2 gene were cloned, their fussion expression vectors were constructed and expressed in 293T cell. The homology of Exon 2 sequence of SUV39H1 gene between Debao pig and sus scrofa was 100%, which was conservative among cattle, sheep and horse. The amino acid sequence of Debao pig SUV39H2 shared 97%,96% and 96% identify with cattle, human and chimpanzee respectively. It demonstrated that the amino acid sequence of SUV39H2 showed high conservation in different species.2. Based on the sequence of siRNA reported, shRNA fragments of SUV39H1/H2 genes were synthesized and inserted into the lentiviral expression vector pSicoR-GFP, named as pSicoR-GFP-SUV39H1 shRNA1/2 and pSicoR-GFP-SUV39H2 shRNAl/2 respectively. The shRNA interference vector and SUV39H1/H2 fusion expression vector were co-transfected into 293T cell at the mass ratio of 3:1, the relative expression level of SUV39H1/H2 genes was analyzed by qRT-PCR. The results revealed that, compared with the negative and blank control groups, the two shRNA fragments had significant inhibition effect on expression of SUV39H1 gene (P<0.05), reached 65.08% and 40.11% respectively. While the expression of SUV39H2 gene was inhibited by pSicoR-GFP-SUV39H2shRNA1/2 remarkably (P<0.05), reached 39.29% and 93.59%. We selected pSicoR-GFP-SUV39H1 shRNA1 and pSicoR-GFP-SUV39H2 shRNA2 in the following experiments.The effects of knockdown SUV39H1/H2 genes on cell growth of Debao pig fibroblastsTo understand the role of SUV39H1/H2 genes inhibition on cell growth of Debao pig fibroblasts, lentivirus containing their shRNAs was obtained by packing three plasmids into 293T using liposomes method. Virus of pSicoR-GFP-SUV39H1 shRNAl and pSicoR-GFP-SUV39H2 shRNA2 was mixed at titer ratio of 1:1, and then added to the cell culture medium with different MOI value (300/600/1200). Cell growth curve was determined by counting the cell number every 24 hours for 8 days. The cells infected with MOI 600 virus were collected and detected by qRT-PCR to analyze the relative expression level of genes coding enzymes of histone modification and cell cyclin. The results revealed that, compared with the blank control group, inhibition of SUV39H1/H2 genes promoted Debao pig fibroblasts proliferation in MOI 300 and 600 groups significantly (P<0.05), Debao pig fibroblasts proliferation infected by MOI 1200 virus was impeded seriously (P<0.05), even though at the logarithmic phase. Results of qRT-PCR analysis confirmed that the knockdown efficiency of SUV39H1/H2 were 64.18% and 59.28%. Compared with the blank control group, the expression of G9a, HDAC1 and DNMT1 genes reduced significantly (P<0.05) in cells infected by MOI 600 virus, while the expression of HAT1 gene increased significantly (P<0.05). The expression of CyclinA2, CyclinB and PCNA genes increased significantly (P<0.05), the expression of CyclinD1 and CyclinD2 genes decreased significantly (P<0.05).The effects of knockdown SUV39H1/H2 genes on development potentiality of pig cloned embryos1. Embryos derived from PA and SCNT were collected to determine the globle levels of H3K9me3 by immunohistochemical staining. The results showed that H3K9me3 was consistently detected during pig preimplantation developmental stages with different patterns. The globle levels of H3K9me3 of embryos derived from PA increased continuously from 1-cell to 8-cell stages, and achieved the highest level at 8-cell stage, it began to immediately decline during the following stages. The globle H3K9me3 level of SCNT embryos was higher than that of PA embryos. In SCNT embryos, H3K9me3 level increased from 1-cell to morula stages, and decreased at blastocyst stage.2. Analysis of expression of gene regulating H3K9me3 during pig early embryonic development. The results showed that the relative expression level of SUV39H1/H2, KDM4D genes in pig preimplantation embryos of SCNT was more abundant than that of PA groups. Their expression levels increased extremely significantly at 2-cell stage (P<0.01). Compared with pig PA embryos, the SETDB1 mRNA transcript profile in SCNT embryos showed a slightly desrease at 2-cell stage (P>0.05), had a significantly increase from 4-cell to morular stages (P<0.05).3. Donor cells were infected by lentivirus with MOI 300 and 600 value, the green cells were selected to inject into the cytoplasm of enucleated egg. Non-infected cell was used as SCNT control. The cleavage rate, the blastocyst rate and the total cell number of blastocyst were analyzed. The results indicated that, compared with the control, the cleavage rate, blastocyst rate and total cell number significantly increased by inhibiting SUV39H1/H2 gene expression (P<0.05). With the increase of virus amount added from MOI 300 to 600, the number of embryo expressing GFP and blastocyst rate increased significantly (P< 0.05), cleavage rate and total cell number of embryos had no significant difference (P> 0.05).The above results indicated that, to some extent, downregulation of SUV39H1/H2 genes could promote growth of Debao pig fibroblasts, and improve the development potentiality of porcine SCNT preimplantation embryos. |
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