Study On Factors Affecting Reprogramming And Development Of Porcine Somatic Cell Nuclear Transfer Embryos

Utilizing of Somatic cell nuclear transfer (SCNT) could rapid copy alarge number of animals with excellent quality and quantity traits. Ourresearch was focused on improving porcine SCNT cloning efficiency,uncovering the impacting factors and underlying molecular cellmechanism on porcine SCNT reprogramming and embryonicdevelopment. Three aspects were included in this study: retrospectivestudy on factors related to embryo recipient and embryos transferred;optimizing culturing system for porcine cloned embryos and searchingout the corresponding mechanism; and production of porcine chimeras.The follow-up data related to cloned pig production collected in ourlaboratory was examined. Spring showed a higher full-term pregnancyrate compared with winter. Abortion was most likely to take placebetween Day27to Day34. Non-ovulating surrogate sows presented ahigher percentage of full-term pregnancies compared with ovulating sows.Based on Life Table Survival Analysis, delivery in normally fertilized andsurrogate sows is expected to be completed before Day117or Day125,respectively. Additionally, the length of pregnancy in surrogate sow wasnegatively correlated with the average litter size.To improve porcine cloning efficiency, SCNT embryos were treatedwith vitamin C, histone deacetylase inhibitor Vaproic acid (VPA) andScriptaid. In addition, the relevant molecular and cellular mechanism wasalso determined. Results showed that the blastocyst-formation rate inSCNT embryos treated with50μg/mL vitamin C for15h after activation (36.0%) was significantly higher than that of untreated SCNT embryos(11.5%). The enhanced in vitro development rate of vitamin C-treatedembryos was associated with an higher Oct4, Sox2and Klf4expressionlevels in blastocysts, as determined by real-time PCR. Treatment with1mM VPA for14to16h following activation significantly increased therate of blastocyst formation of porcine SCNT embryos compared to thecontrol (31.8%vs.11.4%). The cloning efficiency in the treated groupwas significantly improved compared to the control group. We found thattreating SCNT embryos with500nM Scriptaid for15h after activationsignificantly enhanced the blastocyst formation rate (27.7%) comparedwith the untreated group (12.2%). It was also found that treating SCNTembryos with Scriptaid increased the level of acH3K14.Chimeric embryos were generated by aggregating two EGFP-cellderived embryos with two tdTomato-cell derived embryos at the4-cellstage. These SCNT embryos were pre-incubated with1mM VPA for15h.After embryo transfer, live piglets with overt coat color chimeras weresuccessfully generated.