Electroporation and RNA interference in the rodent retina in vivo and in vitro.
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.
Matsuda
Takahiko
T
Department of Genetics and Howard Hughes Medical Institute, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
Cepko
Constance L
CL
D000818:Animals; D030801:Animals, Genetically Modified; D000831:Animals, Newborn; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D018274:Electroporation; D018507:Gene Expression Regulation, Developmental; D049452:Green Fluorescent Proteins; D066298:In Vitro Techniques; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D020419:Photoreceptor Cells, Vertebrate / Q000166:cytology / Q000378:metabolism; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000235:genetics; D012160:Retina / Q000166:cytology / Q000254:growth & development* / Q000378:metabolism*; D014162:Transfection
D018076:DNA, Complementary; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins