条件性修复早期克隆胚胎Xist异常表达提高猪克隆效率研究

Somatic cell nuclear transfer (SCNT) in pigs has developed for more than ten years , however, despite extensive efforts to improve the technique, the cloning efficiency remains low. In mouse SCNT embryos, the expression of Xist, which is responsible for X chromosome inactivation, is upregulated and X chromosome inactivation is aberrant. When Xist was knockout or knockdown by RNAi in the early development stage of SCNT embryos, cloning efficiency cloud be improved about 10 times. Our preliminary study found that the expression of Xist in aborted porcine cloned fetuses was also higher than corresponding gender and embryonic age normal fertilized fetuses. Dysregulation of Xist gene may be responsible for the low efficiency of pig cloning. The researches in cloned mouse revealed that correction of Xist expression only in preimplantation embryos had a long-term effect on their postnatal normality and improved cloning efficiency significantly. So in this project we will study the Xist expression in early development porcine embryos systematically to find out the timeline of Xist aberrant expression in SCNT embryos. Then combine CRISPRi transcription regulation system and Tet-On system to establish a cell line in which the expression of Xist gene can be down regulated by DOX in early development stage of cloned embryos. This cell line will be used as donor cells for somatic nuclear transfer, therefore, in the early development stage of embryos Xist aberrant expression can be corrected by adding DOX and the cloning efficiency is expected to be improved. If the cloning efficiency of pig is improved, the cell line in which Xist expression can be down regulated by DOX can be used for gene modification and then be cloned for gene modified pig production more efficiently.

克隆猪技术虽已有十几年的发展，但克隆效率依然很低。在小鼠研究中发现，小鼠体细胞核移植胚胎X染色体失活异常，X染色体失活相关基因Xist表达上调。当将Xist基因敲除或者以RNAi技术仅在胚胎早期阶段下调Xist表达，即可使小鼠克隆效率提高近十倍。我们前期研究发现在流产的克隆猪胎儿中Xist基因也异常高表达，说明Xist基因的异常表达可能是导致猪克隆效率低的原因。因此在本课题中我们将开展以下研究：1）对猪克隆胚胎早期发育阶段Xist表达及X染色体失活情况进行检测，确定Xist表达异常时间轴；2）将Tet-on系统与CRISPRi基因表达调控系统相结合，筛选获得可在克隆胚胎发育早期阶段条件性下调Xist表达的猪胎儿成纤维细胞系。并对该细胞系进行核移植，根据Xist表达异常时间轴对克隆胚胎发育早期Xist的表达进行修正，以期提高猪克隆效率，使利用体细胞核移植技术进行基因修饰猪制作更加经济、高效。

克隆猪技术虽已有十几年的发展，但克隆效率依然很低。在小鼠研究中发现，小鼠体细胞核移植胚胎X染色体失活异常，X染色体失活相关基因Xist表达上调。敲除Xist基因可使小鼠克隆效率提高近十倍。在本研究中我们系统地比较了猪克隆胚胎和受精胚胎植入前后转录组的差别，发现异常的克隆胚胎出现全染色体水平的基因下调。负责调控X染色体沉默的基因XIST，在猪异常克隆胚胎中显著地高表达。当敲除供体细胞中XIST基因后，可以拯救每条染色体上原本失活或者下调的基因。通过对XIST基因敲除胚胎印记基因表达,DNA甲基化，转绿组和胚胎多能性网络检测分析，发现Xist敲除胚胎与体内正常胚胎更为接近，Xsit敲除后显著提高猪克隆胚胎的发育潜力。将XIST基因敲除的胚胎移植到代孕母体，相对于对照组胚胎，克隆效率提高了约6.9倍。猪克隆效率的提高，将使利用体细胞核移植技术进行基因修饰猪制作更加经济、高效。