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-offunction 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.

Embryonic stem (ES) cells can be maintained in an

In vivo electroporation is a powerful technique for the introduction of genes into organisms. Temporal and spatial regulation of expression of introduced genes, or of RNAi, would further enhance the utility of this method. Here we demonstrate conditional regulation of gene expression from electroporated plasmids in the postnatal rat retina and the embryonic mouse brain. For temporal regulation, Cre/loxP-mediated inducible expression vectors were used in combination with a vector expressing a conditionally active form of Cre recombinase, which is activated by 4-hydroxytamoxifen. Onset of gene expression was regulated by the timing of 4-hydroxytamoxifen administration. For spatial regulation, transgenes were expressed by using promoters specific for rod photoreceptors, bipolar cells, amacrine cells, Mü ller glia or progenitor cells. Combinations of these constructs will facilitate a variety of experiments, including cell-typespecific gene misexpression, conditional RNAi, and fate mapping of progenitor and precursor cells.G ain-of-function and loss-of-function studies using transgenic animals have greatly advanced our understanding of the molecular and cellular mechanisms of development and disease. In particular, conditional gene activation and inactivation have been powerful methods for studies of gene function and for the labeling and manipulation of specific cell populations in vivo (1, 2). Site-specific recombination systems (Cre/loxP and Flp/FRT) are widely used to control gene expression in transgenic mice. By crossing two transgenic lines, one expressing Cre (or Flp) under tissue-specific and/or inducible control and the other carrying two loxP (or FRT) sites, DNA recombination can lead to inducible gene expression or loss of gene expression in restricted tissues at specific times (3). Although such strategies are very useful, they are time-consuming and cannot be applied to species that are difficult to manipulate genetically.In vivo electroporation is a convenient technique for the introduction of genes into a variety of animals, including mouse, rat, and chick (4, 5). We previously reported that plasmid DNAs can be easily delivered to developing mouse/rat retinas by in vivo electroporation (6). The plasmid DNAs, electroporated from the scleral side of the postnatal day 0 (P0) retina, are preferentially introduced into retinal progenitor/precursor cells, which give rise to four different cell types; rod photoreceptors in the outer nuclear layer (ONL), bipolar and amacrine cells in the inner nuclear layer (INL), and Müller glial cells, which extend radial processes spanning the entire thickness of the retina [see supporting information (SI) Fig. 7]. The efficiency of electroporation into the developing postnatal retina is quite good, and transgene expression persists at least for a few months. Moreover, compared with other gene transfer methods, such as viral vectors, in vivo electroporation has several advantages. First, various types of DNA constructs, including RNAi vectors, are readily introduced to th...