Liu Hongbo, Lv Peiru, Zhu Xiangxing, Wang Xianwei, Yang Xiaogan, Zuo Erwei, Lu Yangqing, Lu Shengsheng, Lu Kehuan
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, and College of Animal Science and Technology, Guangxi University, 100 Daxuedong Road, Nanning, 530004, China.
In Vitro Cell Dev Biol Anim. 2014 Oct;50(9):811-21. doi: 10.1007/s11626-014-9776-8. Epub 2014 May 31.
Porcine transgenic cloning has potential applications for improving production traits and for biomedical research purposes. To produce a transgenic clone, kidney fibroblasts from a newborn Guangxi Bama mini-pig were isolated, cultured, and then transfected with red and green fluorescent protein genes using lipofectamine for nuclear transfer. The results of the present study show that the kidney fibroblasts exhibited excellent proliferative capacity and clone-like morphology, and were adequate for generation of somatic cell nuclear transfer (SCNT)-derived embryos, which was confirmed by their cleavage activity and blastocyst formation rate of 70.3% and 7.9%, respectively. Cells transfected with red fluorescent protein genes could be passed more than 35 times. Transgenic embryos cloned with fluorescent or blind enucleation methods were not significantly different with respect to cleavage rates (92.5% vs. 86.8%, p > 0.05) and blastocyst-morula rates (26.9% vs. 34.0%, p > 0.05), but were significantly different with respect to blastocyst rates (3.0% vs. 13.2%, p < 0.05). Cleavage (75.3%, 78.5% vs. 78.0%, p > 0.05), blastocyst (14.1%, 16.1% vs. 23.1%, p > 0.05) and morula/blastocyst rates (43.5%, 47.0% vs. 57.6%, p > 0.05) were not significantly different between the groups of transgenic cloned embryos, cloned embryos, and parthenogenetic embryos. This indicates that long-time screening by G418 caused no significant damage to kidney fibroblasts. Thus, kidney fibroblasts represent a promising new source for transgenic SCNT, and this work lays the foundation for the production of genetically transformed cloned Guangxi Bama mini-pigs.
猪转基因克隆在改善生产性状和生物医学研究方面具有潜在应用价值。为了生产转基因克隆猪，从新生广西巴马小型猪分离出肾成纤维细胞，进行培养，然后使用脂质体转染红色和绿色荧光蛋白基因用于核移植。本研究结果表明，肾成纤维细胞表现出优异的增殖能力和克隆样形态，适合用于生成体细胞核移植（SCNT）胚胎，其卵裂活性和囊胚形成率分别为70.3%和7.9%，证实了这一点。转染红色荧光蛋白基因的细胞可传代超过35次。采用荧光或盲去核方法克隆的转基因胚胎在卵裂率（92.5%对86.8%，p＞0.05）和囊胚-桑椹胚率（26.9%对34.0%，p＞0.05）方面无显著差异，但在囊胚率方面有显著差异（3.0%对13.2%，p＜0.05）。转基因克隆胚胎组、克隆胚胎组和孤雌生殖胚胎组在卵裂率（75.3%、78.5%对78.0%，p＞0.05）、囊胚率（14.1%、16.1%对23.1%，p＞0.05）和桑椹胚/囊胚率（43.5%、47.0%对57.6%，p＞0.05）方面无显著差异。这表明G418长时间筛选对肾成纤维细胞无显著损伤。因此，肾成纤维细胞是转基因SCNT的一个有前景的新来源，这项工作为生产基因转化的克隆广西巴马小型猪奠定了基础。
