激光生物学报, 2023, 32 (3): 0217, 网络出版: 2024-01-17  

斑马鱼 wbp11基因敲除品系的建立

Construction of Zebrafish wbp11 Gene Knockout Line
作者单位
1 湖南师范大学省部共建淡水鱼类发育生物学国家重点实验室, 心脏发育研究中心, 长沙 410081
2 广东省心血管病研究所, 广东省人民医院, 广州 510100广东省心脏病发病机制与精准防治重点实验室, 广州 510100
3 湖南师范大学省部共建淡水鱼类发育生物学国家重点实验室, 心脏发育研究中心, 长沙 410081广东省心脏病发病机制与精准防治重点实验室, 广州 510100
摘要
WBP11是 WW结构域结合蛋白家族的一员, 参与前体 mRNA剪接过程, 是重要的剪接因子。斑马鱼作为重要的模式生物之一, 其基因组中含有人类 WBP11的同源基因 wbp11。为了研究 WBP11在脊椎动物早期发育过程中的作用机制, 利用 CRISPR/Cas9技术构建了斑马鱼 wbp11基因敲除品系。首先, 在 wbp11基因的第二个外显子上设计基因敲除靶位点, 体外转录合成 sgRNA, 通过显微注射技术对斑马鱼进行基因敲除, 得到 F0代嵌合体斑马鱼。随后, 将嵌合体斑马鱼与野生型斑马鱼杂交, 所得到的 F1代斑马鱼进行基因型鉴定, 筛选出其中的杂合子斑马鱼, 并对杂合子斑马鱼的缺失条带进行测序, 结果显示其为 wbp11基因的 2种缺失突变。让同种突变的 F1代杂合子斑马鱼自交, 对得到的后代 F2进行基因型鉴定, 筛选获得了纯合子斑马鱼, 表明 wbp11基因敲除品系构建成功。经显微镜白光成像观察发现, 受精后 48 h的斑马鱼出现心包水肿、心脏线性化、骨骼弯曲畸形, 这可能是由于 wbp11基因突变引起剪接机制异常所导致的。此研究为探究 WBP11基因在脊椎动物的早期发育中的作用机制开辟了道路
Abstract
As a member of the WW domain binding protein family, WBP11 is an important splicing factor involved in the pre-mRNA splicing process. Zebrafish, one of the notable model organisms, contains orthological homologous gene wbp11 in its genome. In order to study the mechanism of WBP11 in the early development in vertebrates, a knockout strain of wbp11 in ze-brafish was constructed using CRISPR/Cas9 technology. Firstly, gene knockout target sites were designed on the second exon
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游诗琦, 陈宇, 杨雪婷, 李韵璇, 乔卿, 罗莹, 姚梅英, 高罗晴, 刘欣, 王跃群, 吴秀山, 李永青. 斑马鱼 wbp11基因敲除品系的建立[J]. 激光生物学报, 2023, 32(3): 0217. YOU Shiqi, CHEN Yu, YANG Xueting, LI Yunxuan, QIAO Qing, LUO Ying, YAO Meiying, GAO Luoqing, LIU Xin, WANG Yuequn, WU Xiushan, LI Yongqing. Construction of Zebrafish wbp11 Gene Knockout Line[J]. Acta Laser Biology Sinica, 2023, 32(3): 0217.

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