Xist敲降可以改善孤雄单倍体囊胚质量

Improving the Quality of Androgenetic Haploid Blastocyst by Xist Knockdown

单倍体胚胎干细胞研究一直以来吸引着研究者们的注意,它可以用作基因修饰的工具或是用于药物筛选。随着孤雄胚胎干细胞系的成功建立,更扩展了单倍体胚胎干细胞的应用前景。但单倍体孤雄胚胎发育率低,胚胎质量差,制约着孤雄单倍体胚胎干细胞的建系。为改善孤雄单倍体胚胎发育潜能及胚胎干细胞建系效率低的问题,我们检测了小鼠(Mus musculus domesticus)孤雄单倍体胚胎的体外发育过程和该过程中Xist基因表达情况。结果发现,孤雄单倍体胚胎囊胚发育率只有10%~14%,发育至囊胚所需时间差异较大,从3.5~5.5 d不等。通过核糖核酸荧光原位杂交实验(RNA-FISH)跟踪Xist基因表达情况,发现其在发育至囊胚阶段的胚胎中呈抑制状态,而在早期胚胎中多呈表达状态。通过si RNA扰低Xist表达,虽然没有改变孤雄单倍体胚胎发育到囊胚的比例,但显著提高了囊胚质量,并提高了接种胚胎内细胞团(ICM)后建立细胞系的效率。结果说明,Xist基因的表达可能是导致小鼠孤雄单倍体胚胎质量差、干细胞建系率低的原因之一。

In mammals, haploidy is normally restricted to the post-meiotic stages of germ line cells and represents the end of cell proliferation, which means that physiological haploidy is incompatible with self-renewal. The successful establishment of androgenetic haploid embryonic stem cell broadened the practical prospect of haploid stem cells, which means, pluripotency, self-renewal, and haploidy can be incorporated together in a single cell line. These haploid ESCs contained only the paternal set of chromosomes and shown pluripotency as well as self-renewal capabilities. Haploid embryonic stem cell had drawn great interest of researchers because of its potential in genetic modification and drug screening.Continuous technological progress in mammalian genetics depended on the availability of mouse embryonic stem cells. Some reverse genetic approaches had taken advantage of the pluripotency of genetically engineered mouse haploid embryonic stem cell to produce animals with germline-transmitted mutations. To generate androgenetic haploid mouse embryos, we performed nuclear transfer （NT） technology, in which a single sperm head from C57/BL6 × DBA/2 F1 mice, instead of a somatic nucleus, was injected into an enucleated oocyte from C57/BL6 × DBA/2 F1 mice. The androgenetic haploid embryo underwent development from 1-cell stage to blastocyst stage （Fig. 1）. Among the 206 haploid embryos that were reconstructed, 30 （14.5%） developed into blastocysts in vitro. After removal of the zona pellucida, blastocysts were cultured in a standard embryonic stem cell （ESC） culture system supplemented with 2i. As the result shown, the unsatisfied blastocyst rate and blastocyst quality is still an obstacle to the derivation of androgenetic haploid stem cells. To improve the quality of androgenetic haploid blastocyst and the efficiency of the androgenetic haploid stem cells derivation, we assessed the expression ofXist gene in the androgenetic haploid embryos during the preimplantation development of mice in vitro. The result showed that the blastocyst rate of androgenetic haploid embryo was 10% - 14% （Table 1）, the time for the development to blastocyst stage varied from 3.5 day to 5.5 day. RNA-FISH results indicated that Xist gene was actively expressed in the early developmental stage, but was silenced in these embryos which developed to blastocyst stage （Fig. 2）. Xist Knockdown by siRNA improved the quality of the blastocyst and the rate of outgrowth when seeded on the feeders, but did not improved the blastocyst rate. These results indicated that the expression ofXist gene is one of the factors for the unsatisfied quality of the androgenetic haploid blastocyst and the low efficency of androgentic haploid stem cell derivation.