Establishment of an efficient electroporation-based knock-in system in chicken primordial germ cells and a rapid method for positive cell selection

Chicken primordial germ cells (PGCs) hold significant value in avian gene-editing breeding and biomedical research. However, existing technologies are plagued by issues such as low transfection efficiency, high costs, and long screening and culture cycles, which have restricted their industrial application. In this study, an optimized electroporation knock-in system for chicken PGCs was established, comprising PGCs, a specific transfection vector (Z-NC_006127.4-200F-U6-TOP1-sgRNA-PGK-Puro-T2A-mCherry-Z-NC_006127.4-200R), Opti-MEM™ transfection buffer, and electroporation parameters (duration of Poring Pause: 10 ms, voltage of Poring Pause: 140 V, and cycles of poring pulses: 6). Additionally, a rapid enrichment method for positive cells based on limiting dilution and puromycin selection was developed. This system improved the transfection efficiency from < 5 % (using liposome transfection) to 54 %, reduced the cost per transfection by 90 %, and enabled the acquisition of positive cell lines with a purity of > 95 % within one month, while the cells retained their stem cell properties. This research provides an efficient and low-cost solution for the industrial application of avian gene-editing technology.