Induction of Germ Cell-like Cells from Dazl-eGFP Knock-In Chicken Somatic Cells via Transgenic Expression of Pluripotency and Germ Cell-Specific Transcription Factors.

Objective: : Germ cell identity is orchestrated by the coordinated action of multiple key transcription factors during embryonic development, involving the induction and regulation of germline-specific gene expression. In chickens, the expression of DEAD-box helicase 4 (DDX4) and Deleted in Azoospermia-like (DAZL) genes plays a pivotal role in germplasm formation and the specification of germ cell lineage from a totipotent genome. This study aimed to investigate the regulatory mechanisms underlying germ cell fate determination.

Methods: : We performed large-scale gene expression profiling to screen and select critical transcription factors. Through this analysis, we identified differentially expressed genes (DEGs) in chicken primordial germ cells (PGCs), including 1,020 transcription factors. Additionally, we generated a chicken DF1 cell line with an enhanced green fluorescent protein (eGFP) reporter precisely knocked into the transcriptional start site of the DAZL gene using the CRISPR-Cas9 system, enabling real-time monitoring of DAZL expression during reprogramming.

Results: : Through analysis of transcription factor binding sites within ~10 kb upstream regions of DDX4 and DAZL, we selected 10 candidate transcription factors for germ cell induction. Subsequently, the 10 transcription factors identified as regulators of germ cell identity were transduced into the DAZL-knock-in eGFP DF1 cells. This approach led to the successful induction of eGFP-expressing cells in vitro, driven by the endogenous DAZL promoter. We further characterized these cells to confirm their germ cell-specific properties.

Conclusion: : Our findings offer new insights into the transcriptional regulation of chicken germ cells by identifying key factors responsible for the activation of DAZL expression. These results present valuable opportunities for advancing germ cell induction from somatic cells, with potential applications of in vitro models for studying germ cell-specific gene regulatory pathways in avian species.