| Handmade cloning (HMC) is a new technology that appears in recent years, which is honoured as "a landmark breakthrough progress" of international cloning and embryonic field. In this paper we optimized the HMC technology system in our lab and explored a new histone deacetylase inhibitor valproic acid (VPA) on the effect of HMC porcine embryonic development potency. The results showed that:1. Optimizing HMC porcine technology system in our laboratory.(1) Improving the denucleated efficiency.Treatment with0.4μg/mL decarburization colchicine (DC) combined with digestion and recovery of3.3mg/mL pronase, DC processing matured oocytes60minutes and pronase recovery25minutes after digestion could acquire the highest denucleated efficiency76.19%, the denucleated efficiency was much hingher in pronase digestion and recovery25min than in15min.(2) Exploring the influence of bonding methods on the fusion efficiency. The denucleated oocytes bonded with oocyte-oocyte-donor and oocyte-donor-oocyte two styles were performed, and oocyte-donor-oocyte showed higher rate of fusion than that of oocyte-oocyte-donor method (87.17%vs74.40%). After the optimization of denucleation and fusion method,36.69%HMC porcine reconstructed embryos developted to blastocyst stage, which was significantly higher than that of before (21.15%).2. Exploring the effect of VPA treatment on developmental competence of HMC porcine reconstructed embryos from different kinds of donor cells.(1) Comparision of the development competency of HMC porcine reconstructed embryo after treatment with different concentration VPA. With granular cell (GC) as donor, the embryos were treatment with VPA for24h. The results showed, the developmental rate of blastocyst at50nmol/L VPA treatment group (48.92%±1.76) was significantly higher than that of0nmol/L,25nmol/L,75nmol/L and100nmol/L VPA treatment groups (36.87%±0.74,32.58%±0.96,37.91%±1.24,32.27%±0.67,P<0.01).(2) Comparision of the effect of different concentration VPA treatment with HMC porcine reconstructed embryo made from PFF for donor. The results indicated, the developmental rate of blastocyst of50nmol/L and75nmol/L VPA treatment group (52.82%±2.50and51.41%±0.85) was much higher than100nmol/L VPA treatment group (45.43%±2.52)(P<0.05), and was also significantly higher than Onmol/L and25nmol/L VPA treatment group (36.69%±0.89and42.95%±2.29, P<0.01)(3) Comparision of the effect of different concentration VPA treatment on HMC porcine reconstructed embryo made from Tr-PFF for donor, the results indicated, the developmental rate of blastocyst at100nmol/L VPA and150nmol/L VPA treatment group (42.74%±1.65and38.57%±0.48) was significantly higher than that of0nmol/L,25nmol/L,50nmol/L and75nmol/L VPA treatment group (32.39%±1.49,31.02%+1.81,28.69%±1.59and33.57%±1.09, P≤0.01).3. Exploring the effect of histone acH3K14level of HMC porcine reconstructed embryos after VPA treatment. The best immunofluorescence staining concentration of primary antibody acH3K14was optimized as1:100, and the second antibody was1:200. We found the H3K14histone acetylation level of2-cell and blastocyst stage reconstructed embryos after50nmol/L VPA treatment (1.044±0.023and1.117±0.020) was significantly higher than the control group (0.913±0.026and1.079±0.017,P<0.01) and parthenogenetic activation group (0.97±0.047and1.026±0.045).VPA treatment with HMC reconstructed embryos24h improved the HMC embryo histone acetylation levels. 4. Determination the expression pattern of development related genes of HMC reconstructed embryos after VPA treatment. The results indicated that:(1) VPA treatment with HMC reconstructed embryos24h could improve the relative expression of HAT1mRNA at1-cell,2-cell and blastocyst stage (3.02±0.04,2.59±0.14and3.83±0.22), which was significantly higher than HMC and PA embryonic HAT1mRNA at1-cell,2-cell and blastocyst stage (2.64±0.07,1.63±0.17,2.73±0.18vs1.00±0.18,1.00±0.26,1.00±0.09)(P<0.01). The VPA treatment also significantly reduced the relative expression of HDAC2mRNA at2-cell and blastocyst stage(0.74±0.04,0.58±0.07)(P<0.01).(2) VPA treatment with HMC reconstructed embryos24h could improve the relative expression of pluripotency genes OCT4at1-cell,2-cell and blastocyst stage (1.30±0.03,1.58±0.17,1.77±0.07) which was significantly higher than that of PA embryonic OCT4mRNA expression level (P<0.01) Comparision with HMC embryo the difference was not significant at the three stages. As for the relative expression of NANOG mRNA, HMC-V embryo was lower than PA embryo at1-cell stage (0.37±0.06VS1.00±0.17)(P<0.01), but it was significantly higher (2.46±0.12,6.15±0.12) than HMC and PA embryo at2-cell and blastocyst stage (1.51±0.09,1.73±0.05VS1.00±0.11,1.00±0.32)(P<0.01).VPA treatment with HMC reconstructed embryos24h, to a certain extent, improved the HMC embryo developmental potential. |
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