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

Overview

Journal Animals (Basel)

Date 2025 Feb 26

PMID 40003070

Authors

Zhifeng Zhao

Xian Zou

Ying Zhu

Yanhua He

Endashaw Jebessa

Jiannan Zhang

Jian Ji

Peng Chen

Chenglong Luo

Affiliations

Soon will be listed here.

Abstract

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 µg of DNA and 100 µL of Entranster-E in 1 × 10 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 () 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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