Ezaki Ryo, Ichikawa Kennosuke, Matsuzaki Mei, Horiuchi Hiroyuki
Laboratory of Immunobiology, Graduate School of Integrated Sciences for Life, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima 739-8528, Japan.
J Poult Sci. 2022 Apr 25;59(2):182-190. doi: 10.2141/jpsa.0210067.
In chickens, primordial germ cells (PGCs) are effective targets for advanced genome editing, including gene knock-in. Although a long-term culture system has been established for chicken PGCs, it is necessary to select a gene-editing tool that is efficient and precise for editing the PGC genome while maintaining its ability to contribute to the reproductive system. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and CRISPR-mediated precise integration into the target chromosome (CRIS-PITCh) methods are superior as the donor vector is easier to construct, has high genome editing efficiency, and does not select target cells, compared to the homologous recombination method, which has been conventionally used to generate knock-in chickens. In this study, we engineered knock-in chicken PGCs by integrating a fluorescent protein gene cassette as a fusion protein into the chicken vasa homolog () locus of chicken PGCs using the CRIS-PITCh method. The knock-in PGCs expressed the fluorescent protein and , facilitating the tracking of PGCs. Furthermore, we characterized the efficiency of engineering double knock-in cell lines. Knock-in cell clones were obtained by limiting dilution, and the efficiency of engineering double knock-in cell lines was confirmed by genotyping. We found that 82% of the analyzed clones were successfully knocked-in into both alleles. We suggest that the production of model chicken from the knock-in PGCs can contribute to various studies, such as the elucidation of the fate of germ cells and sex determination in chicken.
在鸡中，原始生殖细胞（PGCs）是包括基因敲入在内的先进基因组编辑的有效靶点。尽管已经建立了鸡PGCs的长期培养系统，但有必要选择一种高效且精确的基因编辑工具，用于编辑PGC基因组，同时保持其对生殖系统的贡献能力。与传统用于生成敲入鸡的同源重组方法相比，成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关蛋白9（Cas9）和CRISPR介导的精确整合到目标染色体（CRIS-PITCh）方法更具优势，因为其供体载体更易于构建，具有高基因组编辑效率，且无需选择靶细胞。在本研究中，我们使用CRIS-PITCh方法，通过将荧光蛋白基因盒作为融合蛋白整合到鸡PGCs的鸡血管同源物（）基因座中，构建了敲入鸡PGCs。敲入的PGCs表达荧光蛋白和，便于追踪PGCs。此外，我们还对构建双敲入细胞系的效率进行了表征。通过有限稀释获得敲入细胞克隆，并通过基因分型确认构建双敲入细胞系的效率。我们发现，82%的分析克隆在两个等位基因中均成功敲入。我们认为，由敲入PGCs产生的模型鸡可有助于开展各种研究，如阐明鸡生殖细胞的命运和性别决定机制等。
