Electroporation and RNA interference in the rodent retina <i>in vivo</i> and <i>in vitro</i>

TLDR

Genome‑wide studies generate many candidate genes, necessitating rapid functional analysis methods. The study presents a rapid electroporation technique for gain‑ and loss‑of‑function experiments in rodent retina, both in vivo and in vitro. Plasmid DNA injected into the subretinal space of neonatal pups was efficiently taken up by many cells following multiple high‑voltage pulses. The method achieved efficient, minimally damaging transfection of GFP and reporter constructs that persisted for at least 50 days, produced photoreceptor phenotypes via RNAi comparable to knockouts, and worked in both in vivo retina and in vitro explants.

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.