BACKGROUND Somatic cell nuclear transfer (SCNT) is a useful biotechnological tool for transgenic animal production using genetically modified somatic cells (GMSCs). However, there are several limitations preventing successful transgenic animal generation by SCNT, such as obtaining proper somatic donor cells with a sufficiently long life span and proliferative capacity for generating GMSCs. Here, we established simian virus 40 large T antigen (SV40LT)-mediated lifespan-extended canine fibroblast cells (SV40LT-K9 cells) and evaluated their potential as nuclei donors for SCNT, based on cellular integrity and SCNT embryo development. RESULTS SV40LT did not cause canine cell transformation, based on cell morphology and proliferation rate. No anchorage-independent growth in vitro and tumorigenicity in vivo were observed. After SCNT with SV40LT-K9 cells, embryos were transferred into surrogate dogs. All dogs failed to become pregnant. Most embryos did not proceed past the 8-cell stage and only one surrogate showed an implantation trace in its oviduct, indicating that the cells rarely developed into blastocysts. Because of the absence of an in vitro maturation method for canine embryos, we performed identical experiments using porcine fibroblast cells. Similarly, SV40LT did not transform porcine fibroblast cells (SV40LT-Pig cells). During in vitro development of SV40LT-Pig cell-driven SCNT embryos, their blastocyst formation rate was clearly lower than those of normal cells. Karyotyping analysis revealed that both SV40LT-K9 and SV40LT-Pig cells had aberrant chromosomal statuses. CONCLUSIONS Although lifespan-extended canine and porcine cells via SV40LT exhibit no apparent transforming changes, they are inappropriate for use as nuclei donors for SCNT because of their aneuploidy.

中文翻译：

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SV40大T抗原破坏犬和猪体细胞核移植胚胎的胚胎发生。

背景技术体细胞核转移（SCNT）是使用转基因体细胞（GMSC）进行转基因动物生产的有用的生物技术工具。但是，存在一些限制，无法通过SCNT成功地进行转基因动物的产生，例如获得具有足够长的寿命和增殖能力的合适的体细胞供体细胞，以产生GMSC。在这里，我们建立了猿猴病毒40大T抗原（SV40LT）介导的寿命延长的犬成纤维细胞（SV40LT-K9细胞），并根据细胞完整性和SCNT胚胎发育评估了它们作为SCNT的核供体的潜力。结果基于细胞形态和增殖率，SV40LT不会引起犬细胞转化。没有观察到体外的锚定非依赖性生长和体内的致瘤性。在使用SV40LT-K9细胞的SCNT之后，胚胎被转移到代孕犬中。所有的狗都没有怀孕。大多数胚胎没有超过8细胞阶段，只有一个替代物在其输卵管中显示出植入痕迹，这表明这些细胞很少发育成胚泡。由于不存在犬胚胎的体外成熟方法，因此我们使用猪成纤维细胞进行了相同的实验。同样，SV40LT不能转化猪成纤维细胞（SV40LT-Pig细胞）。在SV40LT-Pig细胞驱动的SCNT胚胎的体外发育过程中，它们的胚泡形成率明显低于正常细胞。核型分析表明，SV40LT-K9和SV40LT-Pig细胞均具有异常的染色体状态。结论尽管通过SV40LT延长了寿命的犬和猪细胞没有明显的转化变化，