Achieving Optimal Transfection Conditions in Chicken Primordial Germ Cells Under Feeder- and Serum-Free Medium

文献类型： 外文期刊

第一作者： Zhao, Zhifeng

作者： Zhao, Zhifeng;Zou, Xian;Zhu, Ying;He, Yanhua;Jebessa, Endashaw;Ji, Jian;Chen, Peng;Luo, Chenglong;Zhang, Jiannan

作者机构：

关键词： primordial germ cell; feeder free; electroporation; transfection efficiency; CRISPR/Cas 9

期刊名称：ANIMALS （ 影响因子：2.7； 五年影响因子：3.2 ）

ISSN： 2076-2615

年卷期： 2025 年 15 卷 4 期

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收录情况： SCI

摘要： The successful application of primordial germ cells (PGCs) is an ideal method for generating gene-edited birds. However, barriers to efficient DNA transfection in PGCs lead to low transfection efficiency, limiting the generation of genetically modified chickens. The current study utilized chemical transfection and electroporation methods to determine the optimal transfection conditions for the PGC line under feeder- and serum-free medium. Among the tested methods, the Lonza electroporation system exhibited the highest transduction efficiency, with a previously unreported rate of 71.13 +/- 1.26%. Optimal transfection conditions were achieved using 4 mu g of DNA and 100 mu L of EntransterTM-E in 1 x 106 PGCs. Furthermore, the optimal electroporation conditions resulted in low cell death and normal expression of pluripotency-related genes, highlighting the low cytotoxicity. The resulting electroporation models were then used to deliver the enhanced green fluorescent protein (EGFP) gene to the Z chromosome with a Cas9-gRNA plasmid, achieving a 7-day insertion efficiency of 14.63 +/- 1.07%. Our study highlights the vast potential of electroporation technology for the transfection of PGCs.

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