Two types of transgenic lines for doxycycline-inducible, cell-specific gene expression in zebrafish ultraviolet cone photoreceptors.
Temporal and spatial control of gene expression is important for studying the molecular and cellular mechanisms of development, physiology, and disease. We used the doxycycline (Dox)-inducible, Tet-On system to develop transgenic zebrafish for inducible, cell specific control of gene expression in the ultraviolet (UV) cone photoreceptors. Two constructs containing the reverse tetracycline-controlled transcriptional transactivator (rtTA) gene driven by the UV opsin-specific promoter (opn1sw1) were used to generate stable transgenic zebrafish lines using the Tol2-based transgenesis method. One construct included a self-reporting GFP (opn1sw1:rtTA, TRE:GFP) and the other incorporated an epitope tag on the rtTA protein (opn1sw1:rtTA(flag)). UV cone-specific expression of TRE-controlled transgenes was induced by Dox treatment in larvae and adults. Induction of gene expression was observed in 96% of all larval UV cones within 16 h of Dox treatment. UV cone-specific expression of two genes from a bidirectional TRE construct injected into one-cell Tg(opn1sw1:rtTA(flag)) embryos were also induced by Dox treatment. In addition, UV cone-specific expression of Crb2a(IntraWT) was induced by Dox treatment in progeny from crosses of the TRE-response transgenic line, Tg(TRE:HA-Crb2a(IntraWT)), to the Tg(opn1sw1:rtTA, TRE:GFP) line and the Tg(opn1sw1:rtTA(flag)) line. These lines can be used in addition to the inducible, rod-specific gene expression system from the Tet-On Toolkit to elucidate the photoreceptor-specific effects of genes of interest in photoreceptor cell biology and retinal disease.
Department of Biology, University of Massachusetts, Amherst, MA 01003, United States. Electronic address: mcwest@bio.umass.edu.
Megan C
MC
West
Department of Biology, University of Massachusetts, Amherst, MA 01003, United States. Electronic address: leahc@bio.umass.edu.
Leah J
LJ
Campbell
Department of Biology, University of Massachusetts, Amherst, MA 01003, United States. Electronic address: johnw@bio.umass.edu.
John J
JJ
Willoughby
Department of Biology, University of Massachusetts, Amherst, MA 01003, United States; Molecular and Cellular Biology Program, University of Massachusetts, Amherst, MA 01003, United States. Electronic address: ajensen@bio.umass.edu.
Abbie M
AM
Jensen
D000818:Animals; D030801:Animals, Genetically Modified; D002460:Cell Line; D004318:Doxycycline / Q000494:pharmacology*; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation / Q000187:drug effects*; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics; D009928:Organ Specificity / Q000235:genetics; D020218:Response Elements; D017949:Retinal Cone Photoreceptor Cells / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D015027:Zebrafish / Q000235:genetics* / Q000378:metabolism
D015534:Trans-Activators; D004318:Doxycycline
Conditional expression; Cone photoreceptor; Transgenic zebrafish