In vitro development of porcine transgenic nuclear-transferred embryos derived from newborn Guangxi Bama mini-pig kidney fibroblasts
In vitro development of porcine transgenic nuclear-transferred embryos derived from newborn Guangxi Bama mini-pig kidney fibroblasts
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新生广西巴马小型猪肾成纤维细胞转基因猪胚胎的体外发育
DOI：
10.1007/s11626-014-9776-8
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发表时间：
2014-10-01
影响因子：
2.1
通讯作者：
Lu, Kehuan
中科院分区：
文献类型：
--
作者：
Liu, Hongbo;Lv, Peiru;Lu, Kehuan
猪的转基因克隆在改良生产性状和生物医学研究方面具有潜在的应用前景。为了获得转基因克隆，对新生广西巴马小型猪肾成纤维细胞进行分离、培养，然后利用脂质体将红色和绿色荧光蛋白基因导入细胞核。结果表明，肾成纤维细胞具有良好的增殖能力和克隆形态，适合体细胞核移植(SCNT)胚胎的产生，其卵裂活性和囊胚形成率分别为70.3%和7.9%。转染红色荧光蛋白基因的细胞可传代35次以上。荧光去核法和盲法克隆的转基因胚胎在卵裂率(92.5%vs.86.8%，p>； )和囊胚率(26.9%vs.34.0%，p>； 0.05)方面没有显著差异，但在囊胚率(3.0%vs.13.2%，p<； 0.05)方面差异显著。卵裂率(75.3%，78.5%vs.78.0%，p>； 0.05)、囊胚率(14.1%，16.1%vs.23.1%，p>； 0.05)和桑椹/囊胚率(43.5%，47.0%vs.57.6%，p>； 0.05)在转基因克隆胚组、克隆胚组和孤雌胚组之间无显著差异。这表明G418长时间筛选对肾成纤维细胞无明显损伤。因此，肾脏成纤维细胞是一种很有前途的转基因SCNT的新来源，为生产转基因克隆广西巴马小型猪奠定了基础。
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.