Generation and Characterization of a Transgenic Zebrafish Expressing the Reverse Tetracycline Transactivator

Qilin Gu^{a, b},
Xiaojie Yang^{a, b},
Xiaozhen He^{a, b},
Qing Li^a,
Zongbin Cui^{a
, ,}

a.
The Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
b.
University of Chinese Academy of Sciences, Beijing 100049, China

More Information

Corresponding author: E-mail address: zbcui@ihb.ac.cn (Zongbin Cui)

摘要

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Abstract: Conditional expression of a target gene during zebrafish development is a powerful approach to elucidate gene functions. The tetracycline-controlled systems have been successfully used in the modulation of gene expression in mammalian cells, but few lines of zebrafish carrying these systems are currently available. In this study, we had generated a stable transgenic zebrafish line that ubiquitously expressed the second-generation of reverse Tet transactivator (rtTA-M2). Southern blotting analysis and high-throughput genome sequencing verified that a single copy of rtTA-M2 gene had stably integrated into the zebrafish genome. After induction with doxycycline (Dox), a strong green fluorescent protein (GFP) was seen in rtTA-transgenic eggs injected with pTRE–EGFP plasmids. The fluorescent signal gradually decreased after the withdrawal of Dox and disappeared. However, leaky expression of GFP was undetectable before Dox-induction. Additionally, transgenic embryos expressing rtTA-M2 exhibited no obvious defects in morphological phenotypes, hatching behavior and expression patterns of developmental marker genes, suggesting that rtTA-M2 had little effect on the development of transgenic zebrafish. Moreover, expressed Dickkopf-1 (DKK1) in pTRE-DKK1-injected embryos led to alterations in the expression of marker genes associated with Wnt signaling. Thus, this rtTA-transgenic zebrafish can be utilized to dissect functions of genes in a temporal manner.
- Zebrafish /
- Transgene /
- Tet-on system /
- Reverse Tet transactivator /
- Doxycycline

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参考文献(42)

[1]	Baron, U., Bujard, H. Tet repressor-based system for regulated gene expression in eukaryotic cells: principles and advances Meth. Enzymol., 327 (2000),pp. 401-421
[2]	Belteki, G., Haigh, J., Kabacs, N. et al. Conditional and inducible transgene expression in mice through the combinatorial use of Cre-mediated recombination and tetracycline induction Nucleic Acids Res., 33 (2005)
[3]	Bornkamm, G.W., Berens, C., Kuklik-Roos, C. et al. Stringent doxycycline-dependent control of gene activities using an episomal one-vector system Nucleic Acids Res., 33 (2005)
[4]	Cook, J., McNiven, M., Richardson, G. et al. Aquaculture, 188 (2000),pp. 15-32
[5]	Cronin, C.A., Gluba, W., Scrable, H. The lac operator-repressor system is functional in the mouse Genes Dev., 15 (2001),pp. 1506-1517
[6]	Dupuy, A.J., Akagi, K., Largaespada, D.A. et al. Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system Nature, 436 (2005),pp. 221-226
[7]	Farrer, R.A., Kemen, E., Jones, J.D.G. et al. FEMS Microbiol. Lett., 291 (2009),pp. 103-111
[8]	Feitsma, H., Cuppen, E. Zebrafish as a cancer model Mol. Cancer Res., 6 (2008),p. 685
[9]	Gossen, M., Bujard, H. Tight control of gene-expression in mammalian-cells by tetracycline-responsive promoters Proc. Natl. Acad. Sci. USA, 89 (1992),pp. 5547-5551
[10]	Gossen, M., Freundlieb, S., Bender, G. et al. Transcriptional activation by tetracyclines in mammalian-cells Science, 268 (1995),pp. 1766-1769
[11]	Hasan, M.T., Friedrich, R.W., Euler, T. et al. PLoS Biol., 2 (2004),p. e163
[12]	Huang, C.J., Jou, T.S., Ho, Y.L. et al. Conditional expression of a myocardium-specific transgene in zebrafish transgenic lines Dev. Dyn., 233 (2005),pp. 1294-1303
[13]	Kawakami, K., Shima, A., Kawakami, N. Identification of a functional transposase of the Tol2 element, an Ac-like element from the Japanese medaka fish, and its transposition in the zebrafish germ lineage Proc. Natl. Acad. Sci. USA, 97 (2000),p. 11403
[14]	Kistner, A., Gossen, M., Zimmermann, F. et al. Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice Proc. Natl. Acad. Sci. USA, 93 (1996),pp. 10933-10938
[15]	Knopf, F., Schnabel, K., Haase, C. et al. Dually inducible TetON systems for tissue-specific conditional gene expression in zebrafish Proc. Natl. Acad. Sci. USA, 107 (2010),pp. 19933-19938
[16]	Koponen, J., Kankkonen, H., Kannasto, J. et al. Gene Ther., 10 (2003),pp. 459-466
[17]	Lewandoski, M. Conditional control of gene expression in the mouse Nat. Rev. Genet., 2 (2001),pp. 743-755
[18]	Li, Z., Huang, X., Zhan, H. et al. J. Hepatol., 56 (2012),pp. 419-425
[19]	Livet, J., Weissman, T.A., Kang, H. et al. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system Nature, 450 (2007),pp. 56-62
[20]	Lycett, G.J., Kafatos, F.C., Loukeris, T.G. Genetics, 167 (2004),pp. 1781-1790
[21]	Martinez, R., Estrada, M., Berlanga, J. et al. Growth enhancement in transgenic tilapia by ectopic expression of tilapia growth hormone Mol. Marine. Biol. Biotechnol., 5 (1996),p. 62
[22]	Mo, S., Wang, L., Li, Q. et al. Caveolin-1 regulates dorsoventral patterning through direct interaction with β-catenin in zebrafish Dev. Biol., 344 (2010),pp. 210-223
[23]	Nagano, M., Brinster, C.J., Orwig, K.E. et al. Transgenic mice produced by retroviral transduction of male germ-line stem cells Proc. Natl. Acad. Sci. USA, 98 (2001),p. 13090
[24]	Shinya, M., Eschbach, C., Clark, M. et al. Zebrafish Dkk1, induced by the pre-MBT Wnt signaling, is secreted from the prechordal plate and patterns the anterior neural plate Mech. Dev., 98 (2000),pp. 3-17
[25]	Song, G.L., Li, Q., Long, Y. et al. Effective gene trapping mediated by Sleeping Beauty transposon PloS ONE, 7 (2012),p. e44123
[26]	Stebbins, M.J., Urlinger, S., Byrne, G. et al. Proc. Natl. Acad. Sci. USA, 98 (2001),p. 10775
[27]	Sun, Y.H., Chen, S.P., Wang, Y.P. et al. Biol. Reprod., 72 (2005),pp. 510-515
[28]	Thisse, C., Thisse, B. Nat. Protoc., 3 (2008),pp. 59-69
[29]	Triezenberg, S.J., Kingsbury, R.C., McKnight, S.L. Functional dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression Genes Dev., 2 (1988),pp. 718-729
[30]	Urasaki, A., Morvan, G., Kawakami, K. Genetics, 174 (2006),pp. 639-649
[31]	Urlinger, S., Baron, U., Thellmann, M. et al. Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity Proc. Natl. Acad. Sci. USA, 97 (2000),pp. 7963-7968
[32]	Wang, Z., Fang, B., Chen, J. et al. BMC Genomics, 11 (2010),p. 726
[33]	Westerfield, M.
[34]	Wittbrodt, J., Shima, A., Schartl, M. Medaka – a model organism from the far East Nat. Rev. Genet., 3 (2002),pp. 53-64
[35]	Xiao, D., Sun, Y., Gu, W.W. et al. Tetracycline-controlled transcriptional regulation systems: countermeasures to eliminate basal transgene leaks in Tet-based systems Progr. Nat. Sci., 17 (2007),pp. 11-19
[36]	Yu, H.M.I., Liu, B., Chiu, S.Y. et al. Development of a unique system for spatiotemporal and lineage-specific gene expression in mice Proc. Natl. Acad. Sci. USA, 102 (2005),pp. 8615-8620
[37]	Zerbino, D.R., Birney, E. Genome Res., 18 (2008),pp. 821-829
[38]	Zhou, X., Symons, J., Hoppes, R. et al. Improved single-chain transactivators of the Tet-On gene expression system BMC Biotechnol., 7 (2007),p. 6
[39]	Zhu, P., Narita, Y., Bundschuh, S.T. et al. Optogenetic dissection of neuronal circuits in zebrafish using viral gene transfer and the Tet system Front. Neural. Circuit., 3 (2009),p. 21
[40]	Zhu, Z., He, L., Chen, S. J. Appl. Ichthyol., 1 (1985),pp. 31-34
[41]	Zhu, Z., Ma, B., Homer, R.J. et al. Use of the tetracycline-controlled transcriptional silencer (tTS) to eliminate transgene leak in inducible overexpression transgenic mice J. Biol. Chem., 276 (2001),p. 25222
[42]	Zhu, Z., Zheng, T., Lee, C.G. et al. Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling Semin. Cell Dev. Biol., 13 (2002),pp. 121-128