自从世界首例体细胞克隆绵羊多莉出生以来，体细胞核移植技术已经被广泛应用于各种哺乳动物。牛是克隆动物中最成功的一个种类，尽管如此，这一技术的应用仍然存在流产率高及胎盘、胎儿发育异常等问题。牛克隆胚胎在植入前和植入后早期的丢失率达到70%左右，其中双核滋养层巨细胞发育异常可能在胎盘发育异常中起重要作用。利用含有两个小分子抑制剂(PD0325901和CHIR99021)的培养体系，我们从IVF和SCNT胚胎成功获得可以在外稳定培养和传代的牛类滋养层干细胞系，二者在形态和特性上没有显著差异，但是经基因表达谱芯片分析，二者在表达基因有明显差异。本项目将在前期工作基础上，深入研究AI、IVF和SCNT类滋养层干细胞之间在基因表达、甲基化和分化形成包括双核滋养层巨细胞在内的成熟滋养层的差异，为探索SCNT胚胎胎盘发育异常和预防提供新的思路和策略。

Somatic cell nuclear transfer (SCNT) has been employed to produce clones of a wide variety of mammalian species since the first successful use of differentiated donor cells was reported. However, the success of cloning by nuclear transfer has not come without complication. Fewer than 6% of the reconstructed bovine blastocysts develop into viable offspring. From the time of embryo transfer (day 8) to day 90 of gestation, 70% of SCNT pregnancies are lost. A major problem associated with the mortality of these reconstructed embryos is poor placental development, which has been reported earlier in gestation and during the critical time of early post implantation. The trophoblast binucleate/giant cells (TGC), which makes up approximately 20% of the trophoblast population, and these cells produce pregnancy-related proteins, such as pregnancy-associated glycoproteins (PAGs), placental lactogen (PL), and prolactin-related proteins. Researches indicted that TGC development in SCNT embryos is altered. Given the morphological variation and consequent dysfunction of TGC in placentas from SCNT embryos, it is of considerable interest to explore the expression of factors involved in bovine trophoblast differentiation during the early stages of placental formation. The trophoblast stem cells derived from trophoectderm is the best tool to explore the trophoblast differentiation in vitro. Using a system include two inhibitors (PD0325901 and CHIR99021), we have established bovine trophoblast stem-like cells, which exhibited alkaline phosphatase (AP) activity and expressed pluripotency markers, such as OCT4, NANOG, SOX2, SSEA-1, SSEA-4, TRA-1-60 and TRA-1-81, and trophoblast lineage markers such as CDX2, as determined by both immunofluorescence and RT-PCR. Additionally, these cells generated dome-like structures, formed teratomas when injected into NOD-SCID mice, and differentiated into placenta trophoblast cells in vitro. Based on the preliminary work, this project will further study of the difference in gene expression,methylation status of key maker genens and differentiation potentials between AI、IVF and SCNT derived trophoblast stem-like cells, explore the formation of placenta dysplasia and provide strategies to improve successful rate of SCNT embryos.