猪早期胚胎发育中SOX2基因启动子活性分析

转录因子SOX2(sex determining region Y-box2)在早期胚胎发育第一次谱系分化以及内细胞团多能性维持等方面具有重要的作用。但是目前有关SOX2基因启动子的系统研究较少,尤其是在猪(Sus scrofa)中尚无相关报道。为系统分析猪SOX2基因启动子在早期胚胎中的活性,本研究通过优化显微注射体系中绿色荧光蛋白表达载体种类和注射时间,构建了适合猪SOX2基因启动子活性分析的参照体系和显微注射体系;对猪SOX2基因翻译起始位点上游5000 bp区域进行转录因子结合位点的预测,发现该区域存在4个转录因子结合位点簇;针对上述区域设计并构建相应的缺失型SOX2基因启动子报告载体,利用建立的显微注射体系将其导入胚胎,通过mCherry荧光强度以及qRT-PCR定量分析SOX2基因启动子中不同转录因子结合位点簇对启动子活性的影响。结果表明,与全长SOX2基因启动子相比,SOX2基因翻译起始位点上游2254~2442 bp区域缺失后,猪4-细胞和8-细胞胚胎中SOX2基因启动子活性下降至17.8%,该缺失区域中仅包含两个NF-AT(nuclear factor of activated T cells)转录因子结合位点。因此,本研究结果推测猪SOX2基因启动子中NF-AT转录因子结合位点是影响猪早期胚胎SOX2基因启动子活性的关键位点。本研究为揭示猪早期胚胎发育中SOX2基因表达调控机制提供了数据支持。

Generation and Developmental Characteristics of Porcine Tetraploid Embryos and Tetraploid/diploid Chimeric Embryos

The aim of this study was to optimize electrofusion conditions for generating porcine tet- raploid （4n） embryos and produce tetraploid/diploid （4n/2n） chimeric embryos. Different electric field intensities were tested and 2 direct current （DC） pulses of 0.9 kV/em for 30 p.s was selected as the optimum condition for electrofusion of 2-cell embryos to produce 4n embryos. The fusion rate of 2-cell embryos and the development rate to blastocyst of presumably 4n embryos, reached 85.4% and 28.5%, respectively. 68.18% of the fused embryos were found to be 4n as demonstrated by fluorescent in situ hybridization （FISH）. Although the number of blastomeres in 4n blastocysts was significantly lower than in 2n blastocysts （P 〈 0.05）, there was no significant difference in developmental rates of blastocysts between 2n and 4n embryos （P 〉 0.05）, suggesting that the blas- tocyst forming capacity in 4n embryos is similar to those in 2n embryos. Moreover, 4n/2n chimeric embryos were obtained by aggregation of 4n and 2n embryos. We found that the developmental rate and cell number of blastocysts of 4-cell （4n）/4-cell （2n） chimeric embryos were significantly higher than those of 2-cell （4n）/4-cell （2n）, 4-cell （4n）/8-cell （2n）, 4-cell （4n）/2-cell （2n） chimeric embryos （P 〈 0.05）. Consistent with mouse chimeras, the majority of 4n cells contribute to the trophectoderm （TE）, while the 2n cells are mainly present in the inner cell mass （ICM） of porcine 4n/2n chimeric embryos. Our study established a feasible and efficient approach to produce porcine 4n embryos and 4n/2n chimeric embryos.