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Tracing Transgene Expression in Living Zebrafish Embryos

Köster, Reinhard W. and Fraser, Scott E. (2001) Tracing Transgene Expression in Living Zebrafish Embryos. Developmental Biology, 233 (2). pp. 329-346. ISSN 0012-1606. https://resolver.caltech.edu/CaltechAUTHORS:20160114-101924380

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Abstract

Ectopic expression by injection of plasmid DNA is rarely used in zebrafish embryos due to a low frequency of cells expressing a transgene of interest at detectable levels. Furthermore, the mosaic nature of ectopic expression by plasmid injection requires the direct detection of transgene-expressing cells. We have used the transcriptional activator Gal4-VP16 to amplify transgene expression in living zebrafish embryos. In comparison to conventional expression vectors, Gal4-VP16-amplified expression results in a significant higher number of cells which express a transgene at detectable levels. The Gal4-VP16-activator and the Gal4-VP16-dependent transgene can be placed on a single expression vector. Using tissue-specific regulatory elements, we show that expression of a Gal4-VP16-dependent transgene can be reliably restricted to muscle, notochordal, or neuronal tissues. Furthermore, Gal4-VP16 can drive the expression of two or more transgenes from the same construct resulting in simultaneous coexpression of both genes in virtually all expressing cells. The reported expression system works effectively not only in zebrafish embryos but also in Xenopus embryos, chicken, mouse, and human cultured cells and is thus applicable to a broad variety of vertebrates. The high frequency of transgene expression together with the linked coexpression of more than one transgene opens the possibility of easily monitoring the behavior of individual transgene-expressing cells in real time by labeling them with the fluorescent reporter GFP. The combinatorial nature of the expression system greatly facilitates changing the tissue-specificity, the transgene expressed, or the cell compartment-specific GFP reporter, making it simpler to address a gene's function in different tissues as well as its cell biological consequences.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1006/dbio.2001.0242DOIArticle
http://www.sciencedirect.com/science/article/pii/S0012160601902428PublisherArticle
ORCID:
AuthorORCID
Fraser, Scott E.0000-0002-5377-0223
Additional Information:© 2001 Academic Press. Submitted for publication January 3, 2001. Revised February 8, 2001. Accepted February 11, 2001. Published online April 16, 2001. We thank Helen McBride for continuous discussions and helpful suggestions as well as Ying Gong for her help with zebrafish cell culture. We thank Jed Harmsen, Jennifer Wise, Jon Neri, and Arjun Menon for excellent technical assistance and animal care. We thank Emily Walsh and Didier Stainier for sharing results prior to publication. We thank Kang Shen/Tobias Meyer (pCDNA3-lynGFP), David Stillman (pPC97-VP16), Joseph Dynes/John Ngai (XEX-76/eGFP), Pernille Rorth (pBS 14xUAS), Nico Scheer/José Campos-Ortega (pBSUA-SEIbNotch: intra, pBSGal4), Rusty Lansford (pLZRS.CAH2BEYFP), Virginia Hieber/Daniel Goldman (21696a1TIpEGFP), Shin-ichi Higashijima/Goro Eguchi (a-p-SK), and Shao Jun Du/Randall Moon (pCS-twhh-b-gal-vec) for generously providing plasmids. This work was supported by grants from the Deutsche Forschungsgemeinschaft (to R.W.K.), the Human Frontier Science Program (to R.W.K.), the Beckman Institute, the National Institutes of Health (to S.E.F.), and the National Institute of Mental Health (to S.E.F.).
Funders:
Funding AgencyGrant Number
Deutsche Forschungsgemeinschaft (DFG)UNSPECIFIED
Human Frontier Science ProgramUNSPECIFIED
Caltech Beckman InstituteUNSPECIFIED
NIHUNSPECIFIED
National Institute of Mental Health (NIMH)UNSPECIFIED
Subject Keywords:transgene; Gal4-VP16; UAS; zebrafish; GFP; tissue-specific
Issue or Number:2
Record Number:CaltechAUTHORS:20160114-101924380
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20160114-101924380
Official Citation:Reinhard W. Köster, Scott E. Fraser, Tracing Transgene Expression in Living Zebrafish Embryos, Developmental Biology, Volume 233, Issue 2, 15 May 2001, Pages 329-346, ISSN 0012-1606, http://dx.doi.org/10.1006/dbio.2001.0242. (http://www.sciencedirect.com/science/article/pii/S0012160601902428)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:63679
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:14 Jan 2016 19:16
Last Modified:03 Oct 2019 09:30

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