Clonally derived chicken primordial germ cell lines maintain biological characteristics and proliferative potential in long-term culture

Chicken primordial germ cells (PGCs) are important cells with significant implications in preserving genetic resources, chicken breeding and production, and basic research on genetics and development. Currently, chicken PGCs can be cultured long-term in vitro to produce single-cell clones. However, systematic exploration of the cellular characteristics of these single-cell clonal lines has yet to be conducted. In this study, single-cell clonal lines were established from male and female PGCs of Rugao Yellow Chicken and Shouguang Black Chicken, respectively, using a micropipette-based method for single-cell isolation and culture. Analysis of glycogen granule staining, mRNA expression of pluripotency marker genes (POUV, SOX2, NANOG), germ cell marker genes (DAZL, CVH), and SSEA-1, EMA-1, SOX2, C-KIT, and CVH protein expression showed positive results, indicating that PGCs maintain normal cellular properties after single-cell cloning. Furthermore, tests on proliferation ability and gene expression levels in PGC single-cell clonal lines showed high expression of the pluripotency-related genes and TERT compared to control PGCs, and PGC single-cell clonal lines demonstrated higher proliferation ability. Finally, green fluorescent protein (GFP)-PGC single-cell clonal lines were established, and it was found that these single-cell clonal lines could still migrate into the gonads of recipients, suggesting their potential for germ-line transmission. This study systematically validated the normal cellular characteristics of PGC single-cell clonal lines, indicating that they could be applied in genetic modification research on chickens.