An improved protocol for stable and efficient culturing of chicken primordial germ cells using small-molecule inhibitors
An improved protocol for stable and efficient culturing of chicken primordial germ cells using small-molecule inhibitors
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DOI：
10.1007/s10616-020-00385-9
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发表时间：
2020-02-29
期刊：
影响因子：
2.2
通讯作者：
Horiuchi, Hiroyuki
中科院分区：
文献类型：
--
作者：
Ezaki, Ryo;Hirose, Fumiya;Horiuchi, Hiroyuki
目前，创造转基因鸡最可靠的方法是修改原始生殖细胞（PGCs）的DNA序列。然而，在胚胎发生过程中，只能获得少量的鸡PGCs。因此，体外PGC培养是必要的，以获得足够的细胞用于进一步的基因工程。以前报道的PGC培养方法缺乏通用性。我们在这里报告了一个新的协议，稳定和有效的培养鸡PGCs使用小分子抑制剂。在培养基中加入包括blebbistatin在内的三种小分子抑制剂后，研究了PGCs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PGC的存活率和增殖率增加。Blebbistatin被证明是PGC生长的最有效的诱导剂。与blebbistatin长期培养的PGCs保持典型PGCs的形态，这些细胞表达标记蛋白，如鸡血管同源物（CVH）和NANOG。此外，用荧光蛋白基因转染的PGCs显示出迁移到受体胚胎的性腺中，并且有效地获得了由通过该方法培养的PGCs衍生的后代。这些结果表明，小分子抑制剂代表了稳定和有效的鸡PGC培养的有用工具。
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.