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      Electroporation and RNA interference in the rodent retina in vivo and in vitro

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      Proceedings of the National Academy of Sciences

      Proceedings of the National Academy of Sciences

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          Abstract

          The large number of candidate genes made available by comprehensive genome analysis requires that relatively rapid techniques for the study of function be developed. Here, we report a rapid and convenient electroporation method for both gain- and loss-of-function studies in vivo and in vitro in the rodent retina. Plasmid DNA directly injected into the subretinal space of neonatal rodent pups was taken up by a significant fraction of exposed cells after several pulses of high voltage. With this technique, GFP expression vectors were efficiently transfected into retinal cells with little damage to the operated pups. Transfected GFP allowed clear visualization of cell morphologies, and the expression persisted for at least 50 days. DNA-based RNA interference vectors directed against two transcription factors important in photoreceptor development led to photoreceptor phenotypes similar to those of the corresponding knockout mice. Reporter constructs carrying retinal cell type-specific promoters were readily introduced into the retina in vivo, where they exhibited the appropriate expression patterns. Plasmid DNA was also efficiently transfected into retinal explants in vitro by high-voltage pulses.

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          Most cited references 34

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          Efficient selection for high-expression transfectants with a novel eukaryotic vector

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            Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.

            Mouse genetic manipulation has provided an excellent system to characterize gene function in numerous contexts. A number of mutants have been produced by using transgenic, gene knockout, and mutagenesis techniques. Nevertheless, one limitation is that it is difficult to express a gene in vivo in a restricted manner (i.e., spatially and temporally), because the number of available enhancers and promoters which can confine gene expression is limited. We have developed a novel method to introduce DNA into in/exo utero embryonic mouse brains at various stages by using electroporation. More than 90% of operated embryos survived, and more than 65% of these expressed the introduced genes in restricted regions of the brain. Expression was maintained even after birth, 6 weeks after electroporation. The use of fluorescent protein genes clearly visualized neuronal morphologies in the brain. Moreover, it was possible to transfect three different DNA vectors into the same cells. Thus, this method will be a powerful tool to characterize gene function in various settings due to its high efficiency and localized gene expression.
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              A DNA vector-based RNAi technology to suppress gene expression in mammalian cells.

              Double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful reverse genetic tool to silence gene expression in multiple organisms including plants, Caenorhabditis elegans, and Drosophila. The discovery that synthetic double-stranded, 21-nt small interfering RNA triggers gene-specific silencing in mammalian cells has further expanded the utility of RNAi into mammalian systems. Here we report a technology that allows synthesis of small interfering RNAs from DNA templates in vivo to efficiently inhibit endogenous gene expression. Significantly, we were able to use this approach to demonstrate, in multiple cell lines, robust inhibition of several endogenous genes of diverse functions. These findings highlight the general utility of this DNA vector-based RNAi technology in suppressing gene expression in mammalian cells.
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                Author and article information

                Journal
                Proceedings of the National Academy of Sciences
                Proceedings of the National Academy of Sciences
                Proceedings of the National Academy of Sciences
                0027-8424
                1091-6490
                January 06 2004
                January 06 2004
                November 05 2003
                January 06 2004
                : 101
                : 1
                : 16-22
                Article
                10.1073/pnas.2235688100
                314130
                14603031
                © 2004
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