荧光转基因斑马鱼品系Tg （amh：mCherry）的构建及其性腺发育的示踪分析

doi:10.3969/j.issn.1674-8115.2024.12.012

上海交通大学学报（医学版） ›› 2024, Vol. 44 ›› Issue (12): 1587-1592.doi: 10.3969/j.issn.1674-8115.2024.12.012

• 论著 · 技术与方法 • 上一篇

荧光转基因斑马鱼品系Tg （amh：mCherry）的构建及其性腺发育的示踪分析

吴婧¹(), 徐悦², 凌诗颖², 宋怀东¹, 乔洁²(), 董梅¹()

^1.上海交通大学医学院附属第九人民医院分子诊断科，上海 200011
^2.上海交通大学医学院附属第九人民医院内分泌科，上海 200011

收稿日期:2024-02-19 接受日期:2024-07-18 出版日期:2024-12-24 发布日期:2024-12-24
通讯作者: 乔洁,董梅 E-mail:wujingmirror@163.com;qiaoj2001@126.com;dm20180301@163.com
作者简介:吴婧（1991—），女，初级技师，硕士；电子信箱：wujingmirror@163.com。
基金资助:
国家自然科学基金(82270826)

Construction of fluorescent transgenic zebrafish Tg(amh:mCherry) and tracer analysis of its gonadal development

WU Jing¹(), XU Yue², LING Shiying², SONG Huaidong¹, QIAO Jie²(), DONG Mei¹()

^1.Department of Molecular Diagnostics, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China
^2.Department of Endocrinology, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China

Received:2024-02-19 Accepted:2024-07-18 Online:2024-12-24 Published:2024-12-24
Contact: QIAO Jie,DONG Mei E-mail:wujingmirror@163.com;qiaoj2001@126.com;dm20180301@163.com
Supported by:
National Natural Science Foundation of China(82270826)

摘要/Abstract

摘要：

目的·建立一种用于研究性腺发育及相关疾病的转基因斑马鱼品系Tg（amh:mCherry），并对斑马鱼性腺的发育过程进行示踪分析。方法·使用斑马鱼抗米勒管激素（anti-Müllerian hormone，amh）基因编码区的上游启动子序列和红色荧光蛋白的基因编码序列构建重组转基因表达载体pTol2-amh-mCherry，并采用Sanger测序对构建的载体进行验证。将上述载体和Tol2转座酶的mRNA显微共注射入斑马鱼胚胎，使用荧光显微镜挑选带有荧光的斑马鱼，筛选出能够稳定遗传的转基因斑马鱼品系Tg（amh:mCherry），并通过原位杂交技术检测荧光信号的位置与amh内源性表达位置是否一致。采用荧光显微镜观察F3代转基因斑马鱼的红色荧光情况，并对其性腺发育过程进行追踪和分析。结果·成功构建了pTol2-amh-mCherry载体，并建立了能稳定遗传的F3代转基因斑马鱼品系Tg（amh:mCherry）。原位杂交技术的结果显示，该转基因斑马鱼的红色荧光信号位置与内源性amh表达位置一致。通过对F3代转基因斑马鱼荧光的观察和分析后发现，斑马鱼性腺的发育存在5种情况。结论·Tg（amh:mCherry）转基因斑马鱼品系的成功构建有利于对斑马鱼性腺发育的示踪分析，也为性腺相关疾病的研究提供了较理想的实验模型。

关键词: 抗米勒管激素, Tol2转座酶, 转基因, 斑马鱼, 性腺

Abstract:

Objective ·To establish a transgenic zebrafish line Tg(amh:mCherry) for studying gonadal development and related diseases, and to trace and analyze the developmental process of zebrafish gonads. Methods ·A recombinant transgenic expression vector pTol2-amh-mCherry was constructed by using the upstream promoter sequence of the zebrafish anti-Müllerian hormone (amh) gene coding region and the gene coding sequence of the red fluorescent protein, and was validated by using Sanger sequencing. The recombinant transgenic expression vector and mRNA of Tol2 transposase were co-microinjected into zebrafish embryos, and fluorescent zebrafish were selected by using fluorescence microscope to screen for a stable inherited transgenic zebrafish line Tg(amh:mCherry). In situ hybridization was used to detect whether the location of the fluorescence signals was consistent with the location of endogenous expression of amh. The red fluorescence of transgenic zebrafish from generation F3 was observed by fluorescence microscopy, and the process of gonadal development was tracked and analyzed. Results ·The recombinant transgenic expression vector pTol2-amh-mCherry was successfully constructed, and a stable inherited F3 generation transgenic zebrafish line Tg(amh:mCherry) was successfully established. The results of in situ hybridization showed that the location of the red fluorescence signals in the transgenic zebrafish Tg(amh:mCherry) was consistent with the location of endogenous expression of amh. Through observation and analysis of the fluorescence of transgenic zebrafish from F3 generation, five developmental patterns of zebrafish gonads were found. Conclusion ·The successful construction of Tg(amh: mCherry) transgenic zebrafish line facilitates the tracer analysis of zebrafish gonadal development, which provides a better experimental model for the study of gonad-related diseases.

Key words: anti-Müllerian hormone (amh), Tol2 transposase, transgenesis, zebrafish, gonad

中图分类号:

R-33

吴婧, 徐悦, 凌诗颖, 宋怀东, 乔洁, 董梅. 荧光转基因斑马鱼品系Tg （amh：mCherry）的构建及其性腺发育的示踪分析[J]. 上海交通大学学报（医学版）, 2024, 44(12): 1587-1592.

WU Jing, XU Yue, LING Shiying, SONG Huaidong, QIAO Jie, DONG Mei. Construction of fluorescent transgenic zebrafish Tg(amh:mCherry) and tracer analysis of its gonadal development[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(12): 1587-1592.

图/表 3

图 1 pTol2-amh-mCherry 重组转基因表达载体的构建及其Sanger测序结果Note： A. Construction of pTol2-amh-mCherry recombinant transgenic expression vector. B. Results of Sanger sequencing of amh promoter and mCherry in pTol2-amh-mCherry recombinant transgenic expression vector.

Fig 1 Construction of pTol2-amh-mCherry recombinant transgenic expression vector and its Sanger sequencing results

图 2 F3代转基因斑马鱼的幼鱼、成鱼精巢中 mCherry 的表达位置和内源性 amh 表达位置的一致性分析Note：A. In situ hybridization results of endogenous amh in 30dpf transgenic juvenile zebrafish. B/C. Bright-field observation (B) and mCherry fluorescence observation (C) of 30dpf transgenic juvenile zebrafish. D/E. In situ hybridization results of endogenous amh (D) and mCherry (E) in the spermatheca of adult transgenic zebrafish.

Fig 2 Consistency analysis of mCherry and endogenous amh expression sites in the F3 transgenic juvenile zebrafish and the spermatheca of adult transgenic zebrafish

图3 F3代转基因斑马鱼幼鱼的荧光观察及其性腺发育分析Note： A. Percentage of juvenile zebrafish with fluorescent signals in the total juvenile zebrafish at different time points. B. Five types of gonadal development in the juvenile zebrafish. C. Proportion of juvenile zebrafish with 5 types of gonadal development.

Fig 3 Fluorescence observation and gonadal development analysis of juvenile F3 transgenic zebrafish

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荧光转基因斑马鱼品系Tg （amh：mCherry）的构建及其性腺发育的示踪分析

Construction of fluorescent transgenic zebrafish Tg(amh:mCherry) and tracer analysis of its gonadal development

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