PGC-mediated conservation strategies for germplasm resources of Rugao Yellow Chicken and Shouguang Chicken in China

doi:10.1016/j.jia.2024.05.019

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Guangzheng Liu^{1, 2}, Wenjie Ren^{1, 2}, Kai Jin¹, Dan Zheng^{1, 2}, Qisheng Zuo^{1, 2}, Yani Zhang^{1, 2}, Guohong Chen^{1, 2}, Bichun Li^1,2,#, YingJie Niu^1,#

¹Joint International Research Laboratory of Agriculture and Agri-Product Safety of the Ministry of Education of China, Yangzhou University, Yangzhou 225009, China

²College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China

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摘要【目的】种质资源对地方鸡的生物多样性和畜牧业的可持续发展，以及优质鸡的养殖和产业化至关重要。不幸的是，许多地方和本土鸡品种正面临数量下降的危险，这强调了保护濒危鸡品种种质资源的必要性。原始生殖细胞（PGCs）通过将遗传信息从亲本传递给子代和确保种系间遗传物质的稳定性，对种质资源的保存起着至关重要的作用。本研究旨在探索中国本土鸡通过PGCs的体外培养和冷冻保存进行保护的方法，以促进种质资源的保护和基因编辑鸡的生产。【方法】本研究从鸡胚性腺中分离出PGCs，在无饲养细胞的FAcs培养基中培养建立PGCs系，随后对获得的PGCs系进行鉴定、冻存和活体复原。【结果】通过约40天的持续培养，我们成功建立了多个PGC细胞系，其中包括来自如皋黄鸡和寿光鸡的18个细胞系，建系效率在39.1%~45%之间。PGCs经过40代培养后仍保持典型的PGC特征，糖原染色呈阳性，并同时表达多能性和生殖标记基因。此外，我们发现冷冻保存至120天的PGCs仍保持活力，具有稳定的细胞增殖能力。通过将绿色荧光蛋白（GFP）标记的PGCs注射到受体鸡胚血管中，我们发现这些细胞可以在受体胚胎的生殖腺中发育成配子，从而产生后代。这表明即使经过长时间的培养，PGCs仍保持其迁移和种系传递能力。【结论与创新性】本研究首次成功建立了多个来自不同本地鸡品种的PGC细胞系，并验证了它们在体外培养和冷冻保存条件下的稳定性和功能性。此外，我们还证明了这些PGCs在体内注射后仍能迁移至生殖腺并发育成配子，受体鸡性成熟后成功产生出后代，这为种质资源的保护和转基因鸡生产提供了重要的基础。

Abstract Germplasm resources are essential for the sustainable development of biodiversity and husbandry of local chickens, as well as for the breeding and industry of superior quality chickens. Unfortunately, many local and indigenous chicken breeds are at risk of declining numbers, emphasizing the need to conserve breed resources for endangered chickens. Primordial germ cells (PGCs) are crucial for preserving germplasm resources by inheriting genetic information from parents to offspring and ensuring stability of genetic material between germlines. In this study, PGCs were isolated from chicken embryos' gonads and cultured in FAcs medium without feeder cells. Over a period of approximately 40 days, the cells proliferated to a number of up to 10⁶, establishing various cell lines. Particularly, 18 PGC lines were created from Rugao Yellow Chicken and Shouguang Chicken, with an efficiency ranging from 39.1% to 45%. Furthermore, PGCs that had been cultured for 40 passages exhibited typical PGC characteristics, such as glycogen staining reaction, and expression of pluripotency and reproductive markers. These results confirm that PGCs maintain stem cell properties even after long-term in vitro culture. Additionally, PGCs cryopreserved for up to 120 days remained viable, maintained typical PGC morphologies, and possessed stable cell proliferation ability. Through intravascular injection into chicken embryos, green fluorescent protein (GFP)-PGCs were found in the recipient embryos' gonads and could develop into gametes to produce offspring, indicating that even after extended culture, PGCs retain their migratory and lineage-transmitting capabilities. This research offers valuable insights into the in vitro cultivation and preservation of PGCs of Chinese indigenous chickens. The findings of this study can be applied in transgenic chicken production and the preservation of genetic resources of indigenous chicken breeds.

Keywords: Chicken Primordial germ cell Germplasm resources Cryopreservation

Online: 04 June 2024

Fund: This work was supported by the National Key Research and Development Program of China (2021YFD1200301, 2021YFD1200302), the Natural Science Foundation of Jiangsu Province (BK20210813), the National Natural Science Foundation of China (32102534), and the Yangzhou International Science and Technology Cooperation Projects (YZ2021175).

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Guangzheng Liu, Wenjie Ren, Kai Jin, Dan Zheng, Qisheng Zuo, Yani Zhang, Guohong Chen, Bichun Li, YingJie Niu. 2024. PGC-mediated conservation strategies for germplasm resources of Rugao Yellow Chicken and Shouguang Chicken in China. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2024.05.019

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