卵母细胞核因子对猪克隆胚胎细胞核重编程作用的研究

The low efficiency of reprogramming of the transplanted nuclei is bottleneck in producing cloned pigs or transgenic cloned pigs. Due to enucleation of MⅡoocytes, oocyte nuclei or nuclear factors which may be responsible for somatic reprogramming has been ignored hitherto. Nuclear factors in oocytes may play an key role in somatic reprogramming. In this study, fetal fibroblasts integrated with Oct4-EGFP reporter vector was used as donor cells and injected into intact MⅡoocytes. Following activation , reconstructed oocytes could develop to tetraploid embryos containing nucleis from oocytes and transgenic fetal fibroblasts. We speculate that a combination of maternal factors,nuclear factors and transcription factors into one procedure may improve somatic reprogramming efficiency. In addition , fetal fibroblasts were treated with GV oocyte extract without germinal vesicle..Utilizing the above methods, we investigate the effect of nuclear factors (such as Brg1) on the expression of Oct4 and the developmental competence of embryos. We want to elucidate whether nuclear factors in MⅡoocytes are responsible for somatic reprogramming or not; whether nuclear factors in GV oocytes are responsible for somatic reprogramming or not. The approach outlined here could provide an alternative point of view to investigate the mechanism of somatic reprogramming and scientific evidence for improving the cloning efficiency of pigs.

供体核的重编程效率低下是克隆猪和转基因猪生产过程中的瓶颈与难点。由于核移植技术的缺陷，在众多影响重编程效率的因素中，卵母细胞核因子的重编程作用长期被忽视。卵母细胞核因子可能在重编程过程中发挥关键作用。本研究将整合有Oct4启动子-EGFP报告载体的成纤维细胞核导入未去核的MⅡ期卵母细胞，构建了Oct4-EGFP四倍体胚胎，将母源因子、核因子及转录因子三种重编程因子结合到一种方法中；另外，使用去除生发泡的GV期卵母细胞提取物处理成纤维细胞。.通过上述方法，探讨以Brg1为代表的卵母细胞核因子对供体细胞基因组上Oct4的表达影响及核因子对胚胎发育的影响；阐明MⅡ期卵母细胞核因子及发育后期胚胎中的卵母细胞核因子是否具有重编程作用，同时，阐明GV期卵母细胞核因子对成纤维细胞是否有重编程作用。本研究从卵母细胞核因子的角度探讨体细胞核移植过程中的重编程机制问题，可为提高猪体细胞克隆的效率提供科学依据。

运用转基因体细胞核移植技术生产饲料利用率高、肉质好和抗病能力强的优质猪品系，加快猪的育种进程，是当代畜牧业生产的重要任务。但是，生产克隆动物的总体效率低下，.克隆胚胎体外发育能力差，仍然制约着体细胞核移植技术应用于转基因猪的生产。在MⅡ期卵母细胞中，由卵母细胞核内释放出的核因子物质对核移植重构胚的发育有重要影响，考虑到体细胞核移植的技术特点，也许，正是在MⅡ期卵母细胞去核过程中去除了部分卵母细胞核因子，结果导致克隆胚胎发育异常。在本研究中，将供核细胞融合入未去除核物质的MⅡ期卵母细胞，构建了体细胞核物质和卵母细胞核物质共存的四倍体胚胎，以此作为核移植二倍体胚胎的对照模型。研究结果显示：与未去除核物质的四倍体胚胎比较，核移植二倍体胚胎出现了卵裂迟滞现象；Oct4的重编程过程并不是造成Oct4-EGFP四倍体胚胎、Oct4-EGFP核移植二倍体胚胎发育能力差异的主要原因；影响早期胚胎卵裂的因素决定了Oct4-EGFP四倍体胚胎、Oct4-EGFP核移植二倍体胚胎发育能力的差异。完全生长卵母细胞的核仁前体物质在早期胚胎的卵裂过程中发挥着关键作用，它的存在有助于维持早期胚胎着丝粒卫星DNA序列稳定。之前，大量研究显示：卵母细胞核因子物质在胚胎的早期发育过程中发挥关键作用；现在，我们认为发挥关键作用的核因子物质包括完全生长卵母细胞核仁前体组分。今后，通过研究核移植二倍体重构胚胎中核仁前体物质组装及着丝粒卫星DNA序列的稳定性可为提高克隆胚的卵裂能力，进而为提高体细胞核移植技术的整体效率提供理论基础。