| 慢羽鸡成纤维细胞内源性白血病病毒ev21基因敲除 |
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| 引用本文： | 徐天鹏,吴思乐,温健,韦义荣,李思佳,谢龙,虞霖田,宋中宝,陆阳清.慢羽鸡成纤维细胞内源性白血病病毒ev21基因敲除[J].南方农业学报,2021,52(8):2259-2266. |
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| 作者姓名： | 徐天鹏 吴思乐 温健 韦义荣 李思佳 谢龙 虞霖田 宋中宝 陆阳清 |
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| 作者单位： | 1 广西大学动物科学技术学院/亚热带农业生物资源保护与利用国家重点实验室, 南宁 530005;2 南宁市良凤农牧有限责任公司, 南宁 530005 |
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| 基金项目： | 国家自然科学基金项目（31960157）；广西大学大学生创新创业训练计划项目（201910593306） |
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| 摘 要： | 【目的】探索在慢羽鸡成纤维细胞中敲除ev21基因的可行性，净化鸡群内源性逆转录病毒，同时为快速培育缺失ev21基因的慢羽鸡配套系打下基础。【方法】根据ev21基因序列（KY235336）特点，分别在其5'和3'端各设计2个sgRNA，用于构建4种不同sgRNA的打靶质粒，筛选出在5'和3'端打靶效率较高的sgRNA。然后基于CRISPR/Cas9基因编辑技术对ev21基因进行剪切，并通过同源重组方式以红色荧光蛋白（mCherry）的DNA片段（CAG-mCherry）替换ev21基因，实现对慢羽鸡成纤维细胞内源性白血病病毒ev21基因定点敲除。【结果】在慢羽鸡成纤维细胞中能检测到ev21基因，构建的4种sgRNA（sgRNA1~sgRNA4）均能成功插入对应的打靶质粒中，经嘌呤霉素筛选及T7E1酶切检测，发现转染4种不同sgRNA打靶质粒后慢羽鸡成纤维细胞均有不同程度的死亡，其中又以sgRNA1和sgRNA3的基因敲除效率较高。同时针对同源位点左右同源臂构建表达mCherry的供体质粒，以其转染293T细胞12 h后均能表达出mCherry。以sgRNA1和sgRNA3打靶质粒及供体质粒共同转染慢羽鸡成纤维细胞，观察发现成纤维细胞内的mCherry持续表达，至转染后第30 d通过流式细胞仪分选收集红色荧光阳性成纤维细胞，并提取其总DNA进行PCR鉴定与基因测序，结果显示红色荧光阳性成纤维细胞中有目的片段（CAG-mCherry）插入，即以插入替换方式能实现对ev21基因的敲除。【结论】基于crispr/cas9基因编辑技术的基因敲除方法能成功敲除慢羽鸡成纤维细胞内源性白血病病毒ev21基因，为培育缺失ev21基因的慢羽鸡品系提供技术支持。
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| 关 键 词： | 慢羽鸡 禽白血病病毒(ALV) ev21基因 CRISPR/Cas9 基因敲除 |
| 收稿时间： | 2020-07-20 |
Knockout of endogenous leukemia gene ev21 in slow feathering chicken |
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| XU Tian-peng,WU Si-le,WEN Jian,WEI Yi-rong,LI Si-jia,XIE Long,YU Lin-tian,SONG Zhong-bao,LU Yang-qing.Knockout of endogenous leukemia gene ev21 in slow feathering chicken[J].Journal of Southern Agriculture,2021,52(8):2259-2266. |
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| Authors: | XU Tian-peng WU Si-le WEN Jian WEI Yi-rong LI Si-jia XIE Long YU Lin-tian SONG Zhong-bao LU Yang-qing |
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| Affiliation: | 1 College of Animal Science and Technology, Guangxi University/State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Nanning 530005, China;2 Nanning Liangfeng Agriculture and Animal Husbandry Co., Ltd., Nanning 530005, China |
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| Abstract: | 【Objective】 The objective was to explore the feasibility of knockout of ev21 gene in slow feathering chicken fibroblasts for the endogenous retroviruses purification of chicken flocks, and lay a foundation for rapid breeding of slow feathering chicken lines without ev21 gene.【Method】 Based on the characteristics of ev21 gene sequence(KY235336), two sgRNAs were designed at each of its 5' and 3' ends, respectively, for the construction of four different sgRNA targeting plasmids, and the sgRNAs with higher efficiency at the 5' and 3' ends were screened. Then the ev21 gene was cut based on CRISPR/Cas9 gene editing technology and the ev21 gene was replaced by a DNA fragment of red fluorescent protein(mCherry) (CAG-mCherry) by homologous recombination to achieve targeted knockout of the ev21 gene of endogenous leukemia virus in slow feathering chicken fibroblasts.【Result】 The results showed that the ev21 gene could be detected in slow feathering chicken fibroblasts, and the four constructed sgRNAs(sgRNA1-sgRNA4) could be successfully inserted into the corresponding targeting plasmids. After puromycin screening and T7E1 digestion assay, it was found that slow feathering chicken fibroblasts had different degrees of death after transfection with the four different sgRNAs, among which the knockdown efficiency of sgRNA1 and sgRNA3 was higher. Donor plasmids containing the left and right homology arms and expressing mCherry were also constructed with reference to the homologous site. All 293T cells were able to express mCherry after 12 h transfection with the donor plasmid. Cells were found to consistently express mCherry when slow feathering chicken fibroblasts were co-transfected with sgRNA1 and sgRNA3 targeting plasmids and donor plasmids. Red fluorescent positive fibroblasts were collected by flow cytometry at day 30 after transfection, and their total DNA was extracted for PCR identification and gene sequencing, which showed that the target fragment(CAG-mCherry) was inserted in the red fluorescent positive fibroblasts, and therefore the knockdown of ev21 gene could be achieved by insertion substitution.【Conclusion】 The gene knockout method based on CRISPR/Cas9 gene editing technology can successfully knock out the ev21 gene of endogenous leukemia virus in slow feathering chicken fibroblasts, and provide technical support for breeding slow feathering chicken strains without ev21 gene. |
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