An improved protocol for stable and efficient culturing of chicken primordial germ cells using small-molecule inhibitors

Ezaki Ryo

Hirose Fumiya

Furusawa Shuichi

Horiuchi Hiroyuki

Cytotechnology

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.

Chicken

Primordial germ cells

Small-molecule inhibitor

chicken vasa homolog

NANOG

eng

Springer

This is a post-peer-review, pre-copyedit version of an article published in Cytotechnology. The final authenticated version is available online at: https://doi.org/10.1007/s10616-020-00385-9

This is not the published version. Please cite only the published version. この論文は出版社版ではありません。引用の際には出版社版をご確認、ご利用ください。

[ISSN] 0920-9069

[ISSN] 1573-0778

[DOI] 10.1007/s10616-020-00385-9

[DOI] https://doi.org/10.1007/s10616-020-00385-9