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

Matsuda et al. (2003). Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.2235688100

Retinal gene function relied on slow, size-limited viral vectors and knockout mice; this paper showed that electroporating plasmids into neonatal rodent retinae delivers GFP, promoters and RNAi vectors. Crx and Nrl knockdown matched the knockout phenotypes.

Parts used

EGFPCAG PromoterCMV PromoterEF1α PromoterUBC PromoterIRES

Key findings

  • Subretinal pCAG-GFP plus five 50-ms pulses at P0 labels about 80% of rat and 50% of mouse retinae; expression lasts to P50 and fades after 3–4 weeks.

  • pCAG-GFP electroporated at P0 labels about 80% rods, 15% bipolar cells, 3% Müller glia and 1% amacrine cells at P14, like an MLV retrovirus; horizontal and ganglion cells are unlabeled.

  • EF1α and ubiquitin C promoters out-expressed CMV and CAG in neonatal rat retina, but CMV and EF1α under-label photoreceptors, while ubiquitin C labels like CAG, so promoter choice matters for photoreceptors.

  • Scleral delivery labels 5–20% of P0 mouse explant cells; vitreal delivery, ganglion cells and adult CD1 retinae are poorly transfected, and degenerate adult Swiss-Webster retinae label mostly Müller glia.

  • Electroporated into neonatal rat retina, the rhodopsin (2.2 kb), CABP5 (4.7 kb) and CRALBP (4.0 kb) promoters label photoreceptors, bipolar-like inner nuclear layer cells and Müller glia respectively, as expected.

  • In P0 rat retina, pCAGIG-mRax gave GFP cells with Müller glia or progenitor morphology at P21, whereas empty pCAGIG gave mostly rod photoreceptors, matching the earlier Rax retrovirus result.

  • U6 shRNA vectors against Crx, Nrl and GAPDH cut the matching protein-positive cells among GFP cells at P10; empty U6 did nothing, and U6-Crx and U6-Nrl also lowered rhodopsin.

  • U6-Crx and U6-Nrl cells lacked clear outer segments at P20, as in the knockout mice, and U6-Nrl nuclei sat like cones below the outer limiting membrane; U6-GAPDH cells looked normal.

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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.