Induction of germ cell-like cells from deleted in azoospermia-like enhanced green fluorescent protein gene knock-in chicken somatic cells via transgenic expression of pluripotency and germ cell-specific transcription factors

Abstract

Objective: Germ cell identity is regulated by the coordinated action of multiple key transcription factors during embryonic development, which includes the induction and control of germ-line-specific gene expression. The expression of DEAD-box helicase 4 (DDX4) and deleted in azoospermia-like (DAZL) genes in chickens 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: Large-scale gene expression profiling was conducted to screen and select critical transcription factors. This analysis identified differentially expressed genes in chicken primordial germ cells (PGCs), comprising 1,020 transcription factors. Additionally, we generated a chicken DF1 cell line featuring 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 approximately 10 kb upstream regions of DDX4 and DAZL, resulting in the selection of 10 candidate transcription factors for germ cell induction. Subsequently, the ten 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 conducted further characterization of 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 that activate DAZL expression. These results indicated 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.