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

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-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.

Innate‐like CD8+ T‐cells and NK cells: converging functions and phenotypes

“…CD56 bright NK cells express high levels of cytokine receptors, which enables these cells to produce abundant IFN γ and proliferate in response to cytokines such as IL‐2, IL‐15, IL‐12, IL‐18 and IFN α 48, 53. As CD56 bright NK cells are the dominant NK cell population in tissues including lymph nodes and inflammatory sites, this allows them to interact with DCs and T‐cells at these sites 54, 55, 56, 57. In contrast, CD56 bright NK cells secrete little IFN γ in response to target cell recognition mediated by receptors such as NKG2D, even though the receptor is equally expressed by CD56 bright and CD56 dim cells 58.…”

Optogenetic therapy for retinitis pigmentosa

“…In the healthy retina, amacrine cell activity is important for a variety of retinal functions, for example, for the detection of various components of image or eye motion. Using in vivo electroporation, 50 a method that is only feasible in experimental animals, ChR2 has been targeted to ON bipolar cells of rd1 mice. In these mice, the ON ganglion cells were activated by ChR2-expressing ON bipolar cells, and inhibited by amacrine cells, which were themselves excited by the light-sensitive ON bipolar cells.…”

Cigarette smoke up‐regulates PDE3 and PDE4 to decrease cAMP in airway cells

“…Therefore, biophysical methods, including FRET‐based biosensors, have been developed to facilitate real‐time measurement. FRET allows the visualization of cAMP fluctuations in living cells with high temporal and spatial resolution (Adams et al ., 1991; DiPilato et al ., 2004; Nikolaev et al ., 2004; Violin et al ., 2008; Sprenger et al ., 2015). Although FRET has previously been used to estimate cAMP levels in human airway epithelial cells (Schmid et al ., 2006, 2015), airway smooth muscle cells (Billington and Hall, 2011) and endothelial cells (Yañez‐Mó et al ., 2008), real‐time monitoring cAMP levels in the lung tissue has not been reported so far.…”