At present, the most reliable method for creating genetically modified chickens is the modification of the DNA sequence of primordial germ cells (PGCs). However, during embryogenesis, only a small number of chicken PGCs can be obtained. Therefore, in vitro PGC culturing is necessary to obtain sufficient cells for further genetic engineering. Previously reported PGC culturing methods lack versatility. We report here a new protocol for stable and efficient culturing of chicken PGCs using small-molecule inhibitors. The growth rate of PGCs was investigated following the addition of three small-molecule inhibitors, including blebbistatin, into the culture medium. Chicken PGC survival and proliferation rates increased after the addition of small-molecule inhibitors, compared with the untreated control. Blebbistatin was shown to be the most effective inducer of PGC growth. Long-term culturing of PGCs with blebbistatin maintained the morphology of typical PGCs, and these cells expressed marker proteins such as chicken vasa homolog (CVH) and NANOG. Additionally, PGCs transfected with a fluorescent protein gene were shown to migrate into the gonads of the recipient embryo, and progeny derived from PGCs cultured by this method were efficiently obtained. These results demonstrate that small-molecule inhibitors represent a useful tool for stable and efficient chicken PGC culturing.

中文翻译：

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使用小分子抑制剂稳定有效地培养鸡原始生殖细胞的改进方案。

目前，生产转基因鸡的最可靠方法是对原始生殖细胞（PGC）DNA序列的修饰。但是，在胚胎发生过程中，只能获得少量的鸡PGC。因此，体外PGC培养对于获得足够的细胞用于进一步的基因工程是必要的。先前报道的PGC培养方法缺乏通用性。我们在这里报告了一种使用小分子抑制剂稳定高效培养鸡肉PGC的新方案。在向培养基中添加三种小分子抑制剂（包括抑菌素）后，研究了PGC的生长速率。与未处理的对照组相比，添加小分子抑制剂后，鸡的PGC存活率和增殖率增加。胆囊抑素被证明是最有效的PGC生长诱导剂。用blebbistatin长期培养PGC可以保持典型PGC的形态，并且这些细胞表达标记蛋白，例如鸡vasa同源物（CVH）和NANOG。另外，显示出用荧光蛋白基因转染的PGC迁移到受体胚胎的性腺中，并有效地获得了通过这种方法培养的PGC的后代。这些结果表明，小分子抑制剂代表了稳定有效的鸡PGC培养的有用工具。显示出用荧光蛋白基因转染的PGC迁移到受体胚胎的性腺中，并有效地获得了通过该方法培养的PGC的后代。这些结果表明，小分子抑制剂代表了稳定有效的鸡PGC培养的有用工具。显示出用荧光蛋白基因转染的PGC迁移到受体胚胎的性腺中，并有效地获得了通过该方法培养的PGC的后代。这些结果表明，小分子抑制剂代表了稳定有效的鸡PGC培养的有用工具。