Purchase this article with an account.
S. Yoshikawa, X. C. Zhao; PhiC31 Integrase-Based Targeted Cell Ablation System in Zebrafish. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4503.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To study the function of ocular tissues and their interactions in the zebrafish eye, we developed a novel cell ablation system in which an inactive diphtheria toxin A (DTA) gene can be activated by inversion mediated by a bacteriophage-derived DNA recombinase PhiC31 integrase (C31int).
The non-toxic mCerulean (mCer) construct and the DTA construct were designed to test if C3lint converts the inactive DTA to active DTA for cell ablation. To inactive DTA, two artificial exons of the DTA coding region were separated by an artificial intron in the opposite direction of the ubiquitous cytoplasimic actin promoter and flanked by the C31int target sites attP and attB. After an inverse recombination at these target sites by C31int, DTA is activated and cells expressing DTA are ablated. The mCer construct was similarly generated. These constructs were co-injected into the one-cell embryos with C31int RNA or the negative control SB100X RNA and mCer expression and embryos were observed and compared.
Co-injection of the mCer construct and C31int RNA showed 90% mCer-positive embryos (N=71) while no embryos were positive with the control RNA (N=20), indicating a high efficient mCer reconstruction by C31lint. When the DTA construct was co-injection with C31int RNA, 76% of the embryos died in 24 hr and only 4% of the embryos developed normally (N=121), demonstrating effective cell ablation by the reconstructed DTA. When co-injected with the control RNA, the majority of embryos (70%) developed normally (N=92), which was significantly higher than that with C31lint (4%) and slightly lower than the uninjected embryos from the same batch (88%, N=51), suggesting a minimum toxicity of the inactive DTA.
The C31int-based inducible transgenic system can efficiently convert an inactive gene into a functional protein in zebrafish. The minimum toxicity of the inactive DTA and its high efficient conversion into active DTA by C31lint indicate that stable transgenic lines can be easily established and inducible targeted cell ablation can be efficiently achieved. Combined with tightly controlled ocular cell type-specific C31int transgenic zebrafish, targeted cell ablation in specific ocular cells or tissues is possible in the lens, cornea, annular ligament, retinal ganglion cells, and photoreceptors.
This PDF is available to Subscribers Only