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.

IF 2.5 2区 农林科学 Q1 AGRICULTURE, DAIRY & ANIMAL SCIENCE
Bo Ram Lee, Hyeon Yang, Sun Keun Jung, Sung June Byun, Tae Sub Park
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引用次数: 0

Abstract

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.

通过多能性和生殖细胞特异性转录因子的转基因表达诱导Dazl-eGFP敲入鸡体细胞的生殖细胞样细胞
目的:生殖细胞身份是由胚胎发育过程中多个关键转录因子的协同作用精心安排的,涉及生殖系特异性基因表达的诱导和调控。在鸡中,DEAD-box解旋酶4 (DDX4)和Deleted In azoosperia -like (DAZL)基因的表达在种质形成和生殖细胞谱系的形成中起着关键作用。本研究旨在探讨生殖细胞命运决定的调控机制。方法:采用大规模基因表达谱法筛选关键转录因子。通过这一分析,我们鉴定了鸡原始生殖细胞(PGCs)中的差异表达基因(deg),包括1,020个转录因子。此外,我们使用CRISPR-Cas9系统生成了一个鸡DF1细胞系,该细胞系将增强型绿色荧光蛋白(eGFP)报告基因精确敲入DAZL基因的转录起始位点,从而能够在重编程过程中实时监测DAZL的表达。结果:通过分析DDX4和DAZL上游~ 10kb区域的转录因子结合位点,筛选出10个生殖细胞诱导的候选转录因子。随后,10个被鉴定为生殖细胞身份调节因子的转录因子被转导到dazl敲入的eGFP DF1细胞中。这种方法在体外成功诱导了由内源性DAZL启动子驱动的egfp表达细胞。我们进一步对这些细胞进行了表征,以确认它们的生殖细胞特异性。结论:我们的研究结果通过确定DAZL表达激活的关键因子,为鸡生殖细胞的转录调控提供了新的见解。这些结果为推进体细胞生殖细胞诱导提供了宝贵的机会,并有可能应用于研究鸟类生殖细胞特异性基因调控途径的体外模型。
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来源期刊
Animal Bioscience
Animal Bioscience AGRICULTURE, DAIRY & ANIMAL SCIENCE-
CiteScore
5.00
自引率
0.00%
发文量
223
审稿时长
3 months
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