外源移植PGCs通过诱导不育鸡宿主高效生殖系遗传

doi:10.1016/j.jia.2024.08.009

Journal of Integrative Agriculture ›› 2026, Vol. 25 ›› Issue (1): 227-234.DOI: 10.1016/j.jia.2024.08.009

外源移植PGCs通过诱导不育鸡宿主高效生殖系遗传

收稿日期:2024-02-21 修回日期:2024-08-20 接受日期:2024-07-16 出版日期:2026-01-20 发布日期:2025-12-09

Establishing an induced infertile chicken line for efficient germline transmission of exogenous PGCs

Haimei Qin^{1, 2*}, Xiaoxuan Jia^1*, Zhenwen Huang¹, Yifei Zhi¹, Na Ji¹, Meiyu Lan¹, Lang Zhang¹, Xingting Liu¹, Huiyan Xu¹, Yangqing Lu^1#

¹Guangxi Key Laboratory of Animal Breeding, Disease Control and Prevention, College of Animal Science and Technology, Guangxi University, Nanning 530004, China
²Youjiang Medical University for Nationalities, Baise 533000, China

Received:2024-02-21 Revised:2024-08-20 Accepted:2024-07-16 Online:2026-01-20 Published:2025-12-09
About author:Haimei Qin, E-mail: haimeiqin5@163.com; Xiaoxuan Jia, E-mail: 2118391013@st.gxu.edu.cn; #Correspondence Yangqing Lu, Tel: +86-771-3274214, E-mail: lyq@gxu.edu.cn
Supported by:
This work was supported by the National Key R&D Program of China (2021YFD1300100), Guangxi Key R&D Program, China (AB21220005), Reproductive Medicine, Guangxi Medical and Health Key Discipline Construction Project of the Affiliated Hospital, and the National Natural Science Foundation of China (32360180).

摘要/Abstract

摘要：

目的利用自杀基因构建一种可诱导清除内源性PGCs的新型不育鸡，以作为外源的原始生殖细胞（Primordial germ cells，PGCs）移植的宿主，提升生殖系遗传效率。方法研究采用CRISPR-Cas9基因编辑方法，将单纯疱疹病毒胸苷激酶（Herpes simplex virus thymidine kinase, HSV-TK）自杀基因定点敲入DAZL基因最后一个外显子，构建DAZL-HSV-TK-EGFP PGCs，通过鸡胚移植制备HSV-TK不育鸡，并在HSV-TK鸡胚中添加更昔洛韦（Ganciclovir, GCV）消除内源生殖细胞后，移植体外培养的PGCs及转基因mCherry_PGCs，制备生殖系嵌合体鸡，以实现外源PGCs在HSV-TK不育鸡中高效率的生殖遗传效率。结果本研究体外试验显示，添加10 μM浓度的GCV底物能够在体外培养条件下诱导DAZL-HSV-TK-EGFP PGCs凋亡；动物试验显示，HSV-TK鸡胚中观察到EGFP阳性PGCs，孵化成熟的公鸡睾丸中存在生精细胞，但是在鸡胚中加入100 µL的浓度为0.165 mM的GCV后，鸡胚PGCs被消除，出生后的公鸡睾丸仍含有完整的生精小管，但没有生精细胞；同样，GCV处理后孵化在获得的母鸡卵巢中未观察到卵泡。以添加GCV之后的HSV-TK鸡胚作为宿主，移植外源mCherry PGCs后发现，鸡胚内源性的EGFP PGCs细胞被消除，仅观察到移植的红色荧光的mCherry PGCs；制备获得嵌合体孵化成熟后，其配种后代均未检测到HSV-TK基因，说明来自外源移植的生殖细胞。结论本研究成功构建了一种可诱导消除内源生殖细胞的型不育鸡，利用其鸡胚作为外源PGCs细胞移植宿主，能直接提高了外源PGCs移植后的生殖系遗传效率。该不育鸡在种质资源保护和基因编辑方面具有重要的应用价值。创新性 本研究首次利用HSV-TK细胞自杀消融系统制备获得可诱导消除内源性PGCs的新型不育鸡，作为外源PGCs移植的宿主实现高效的生殖系遗传。

Abstract:

Primordial germ cells (PGCs) are the stem-cell population of adult animal gametes, which develop into sperm or eggs. It can be propagated in vitro and injected into the host chicken for genome editing to obtain germline chimeric chicken. However, it has the limitation that the host embryo contains endogenous PGCs, which raises complications, resultantly donor PGCs fail to compete, and transmission efficiency reduced. Therefore, to increase the transmission efficiency, we generated a novel sterile chicken with the inducible elimination of endogenous PGCs in the host. This is the first study that applied the herpes simplex virus thymidine kinase (HSV-TK) cell ablation system in avian. CRISPR/Cas9-mediated homology-directed repair was performed to localize the HSV-TK suicide gene to the last exon of the deleted in azoospermia-like (DAZL) gene, and ganciclovir (GCV) was added to induce the apoptosis in the germ cells of the host embryo. The sterilized host embryo introduced genome-edited PGCs to produce chimeric chicken carrying exogenous germ cells only. It was observed that the germline transmission efficiency was 100% achieved, and the obtained chicks were purely from donor breeds. The technologies established in the current study have important applications in germplasm conservation and gene editing in chicken.

Key words: chicken embryos , germline transmission efficiency , herpes simplex virus thymidine kinase , primordial germ cells

Haimei Qin, Xiaoxuan Jia, Zhenwen Huang, Yifei Zhi, Na Ji, Meiyu Lan, Lang Zhang, Xingting Liu, Huiyan Xu, Yangqing Lu. 外源移植PGCs通过诱导不育鸡宿主高效生殖系遗传[J]. Journal of Integrative Agriculture, 2026, 25(1): 227-234.

Haimei Qin, Xiaoxuan Jia, Zhenwen Huang, Yifei Zhi, Na Ji, Meiyu Lan, Lang Zhang, Xingting Liu, Huiyan Xu, Yangqing Lu. Establishing an induced infertile chicken line for efficient germline transmission of exogenous PGCs[J]. Journal of Integrative Agriculture, 2026, 25(1): 227-234.

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外源移植PGCs通过诱导不育鸡宿主高效生殖系遗传

Establishing an induced infertile chicken line for efficient germline transmission of exogenous PGCs

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