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Jody A. Summers Rada, Lindsey R. Hollaway; Development of Multicistronic Lentiviral Vectors for In Vivo Studies on Postnatal Ocular Growth in Chicks. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3447.
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In order to assess the functional role of specific genes during the process of emmetropization, we designed an inducible lentiviral expression vector and developed a method of gene delivery in ovo to enable inducible gene expression in the choroid and/or sclera of posthatch chicks for the potential control of postnatal ocular growth.
We constructed lentiviral vectors modified from the pINDUCER series of expression vectors that allow conditional expression of the retinoic acid synthesizing enzyme, RALDH2, and DSred under the regulation of the TRE2 promoter and ubiquitous expression of eGFP under the constituitive EF1α promoter. 293T cells were transduced with lentiviral constructs and viral transduction was monitored by expression of eGFP. Following treatment of 293T cells with doxycycline (1ug/ml) RALDH2 and DSred expression were assessed by DSred fluorescence, immunocytochemistry and western blotting using specific anti-RALDH2 antibodies. For in vivo studies, a lentiviral vector expressing DSred under the regulation of a CMV promoter was microinjected to the blastoderm of 1 day old chick embryos and eggs were incubated to hatching. Viral transduction was evaluated by DSred fluorescence and by PCR detection of the DSred transgene in several chick tissues.
Infection of 293T cells with our conditional TRE2-RALDH2 lentiviral construct resulted in constitutive eGFP fluorescence in a dose-dependent manner. Doxycycline treatment resulted in inducible DSred fluorescence and RALDH2 protein expression in all transduced cells. Following in ovo injection of the CMV-DSred lentiviral construct, the presence of a 700 bp DSred DNA PCR product was detected in skin, brain, heart, and whole eye of posthatch chicks. Additionally, DSred fluorescence was detected in the retina, choroid and sclera.
Our lentiviral vector provides an efficient method for inducible RALDH2 protein expression as well as fluorescent detection of viral transduction and gene induction. Moreover, in ovo delivery of lentiviral vectors to chick embryos results in transgene expression in a variety of ocular and extraocular chick tissues. These studies provide the foundation for the development of transgenic chickens that conditionally express RALDH2 in ocular and extraocular tissues.
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