Journal of Shanghai Jiao Tong University (Medical Science) >

2024 , Vol. 44 >Issue 12: 1587 - 1592

DOI: https://doi.org/10.3969/j.issn.1674-8115.2024.12.012

Techniques and methods

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

  • Jing WU ,
  • Yue XU ,
  • Shiying LING ,
  • Huaidong SONG ,
  • Jie QIAO ,
  • Mei DONG
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  • 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
QIAO Jie, E-mail: qiaoj2001@126.com.
DONG Mei, E-mail: dm20180301@163.com

Received date: 2024-02-19

  Accepted date: 2024-07-18

  Online published: 2024-12-24

Supported by

National Natural Science Foundation of China(82270826)

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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

Cite this article

Jing WU , Yue XU , Shiying LING , Huaidong SONG , Jie QIAO , Mei DONG . 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 . DOI: 10.3969/j.issn.1674-8115.2024.12.012

References

1 YE M L, CHEN Y. Zebrafish as an emerging model to study gonad development[J]. Comput Struct Biotechnol J, 2020, 18: 2373-2380.
2 FONTANA B D, MEZZOMO N J, KALUEFF A V, et al. The developing utility of zebrafish models of neurological and neuropsychiatric disorders: a critical review[J]. Exp Neurol, 2018, 299(Pt A): 157-171.
3 ROSA J G S, LIMA C, LOPES-FERREIRA M. Zebrafish larvae behavior models as a tool for drug screenings and pre-clinical trials: a review[J]. Int J Mol Sci, 2022, 23(12): 6647.
4 BAMBINO K, CHU J. Zebrafish in toxicology and environmental health[J]. Curr Top Dev Biol, 2017, 124: 331-367.
5 SUN D, ZHANG Y, WANG C, et al. Sox9-related signaling controls zebrafish juvenile ovary-testis transformation[J]. Cell Death Dis, 2013, 4(11): e930.
6 GIANONCELLI A, GUARIENTI M, FRAGNI M, et al. Adrenocortical carcinoma xenograft in zebrafish embryos as a model to study the in vivo cytotoxicity of abiraterone acetate[J]. Endocrinology, 2019, 160(11): 2620-2629.
7 HINFRAY N, NóBREGA R H, CAULIER M, et al. Cyp17a1 and Cyp19a1 in the zebrafish testis are differentially affected by oestradiol[J]. J Endocrinol, 2013, 216(3): 375-388.
8 PFENNIG F, STANDKE A, GUTZEIT H O. The role of Amh signaling in teleost fish: multiple functions not restricted to the gonads[J]. Gen Comp Endocrinol, 2015, 223: 87-107.
9 BAUTISTA F E A, VARELA JUNIOR A S, CORCINI C D, et al. The herbicide atrazine affects sperm quality and the expression of antioxidant and spermatogenesis genes in zebrafish testes[J]. Comp Biochem Physiol C Toxicol Pharmacol, 2018, 206/207: 17-22.
10 WU K, SONG W Y, ZHANG Z W, et al. Disruption of dmrt1 rescues the all-male phenotype of the cyp19a1a mutant in zebrafish:a novel insight into the roles of aromatase/estrogens in gonadal differentiation and early folliculogenesis[J]. Development, 2020, 147(4): dev182758.
11 CATE R L, MATTALIANO R J, HESSION C, et al. Isolation of the bovine and human genes for Müllerian inhibiting substance and expression of the human gene in animal cells[J]. Cell, 1986, 45(5): 685-698.
12 JOSSO N, BELVILLE C, CLEMENTE N D, et al. AMH and AMH receptor defects in persistent Müllerian duct syndrome[J]. Hum Reprod Update, 2005, 11(4): 351-356.
13 YAN Y L, BATZEL P, TITUS T, et al. A hormone that lost its receptor: anti-Müllerian hormone (AMH) in zebrafish gonad development and sex determination[J]. Genetics, 2019, 213(2): 529-553.
14 RODRíGUEZ-MARí A, YAN Y L, BREMILLER R A, et al. Characterization and expression pattern of zebrafish anti-Müllerian hormone (Amh) relative to sox9a, sox9b, and cyp19a1a, during gonad development[J]. Gene Expr Patterns, 2005, 5(5): 655-667.
15 KAWAKAMI K, KOGA A, HORI H, et al. Excision of the tol2 transposable element of the medaka fish, Oryzias latipes, in zebrafish, Danio rerio[J]. Gene, 1998, 225(1/2): 17-22.
16 THISSE C, THISSE B. High-resolution in situ hybridization to whole-mount zebrafish embryos[J]. Nat Protoc, 2008, 3(1): 59-69.
17 ZHU J J, ZHANG D W, LIU X, et al. Zebrafish prmt5 arginine methyltransferase is essential for germ cell development[J]. Development, 2019, 146(20): dev179572.
18 ORBAN L, SREENIVASAN R, OLSSON P E. Long and winding roads: testis differentiation in zebrafish[J]. Mol Cell Endocrinol, 2009, 312(1/2): 35-41.
19 PAN Y J, TONG S K, HSU C W, et al. Zebrafish establish female germ cell identity by advancing cell proliferation and meiosis[J]. Front Cell Dev Biol, 2022, 10: 866267.
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