Purchase this article with an account.
Bikash R Pattnaik, Pawan K Shahi, Bryce Aul, Akshita Pattnaik, Yu Chang, De-Ann M Pillers; Mouse Retina after Kcnj13 Inhibition by RNAi is a Model for LCA and SVD. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5880.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
The inwardly rectifying potassium ion-channel Kir7.1, is present in the apical aspect of RPE cells. Isolated RPE cell electrophysiology demonstrate a robust Kir7.1 channel current. When the gene KCNJ13 is mutated, it results in a dysfunctional channel and childhood blindness. We sought to study the loss of Kcnj13 gene function in mice using RNAi knock down of Kcnj13 in order to overcome the embryonic lethality in a conventional knockout.
We followed the ARVO statement for the use of animals. Adult 6 wk old C57Bl6 mice were anesthetized with ketamine (80 mg/kg) and xylazine (16 mg/kg). Kir7.1 shRNA or control shRNA-GFP lentiviral particles (2 µl) were delivered to vitreous using a UMP3 NanoFil (WPI, Sarasota, FL). Fifteen days post injection, isolated RPE cells were used to determine transcript expression. Mice dark-adapted overnight before injection, or 2, 4, 7, or 14 days post-injection of shRNA underwent electroretinography. Both the a- and b-waves under scotopic conditions were measured in response to 0.1 to 100 cd.s/m2 light flash. The c-wave at the higher light flash intensities and the response to NaN3 by tail vein injection was carried out to specifically test RPE function.
Kcnj13 transcripts were not detected after shRNA knock down. The ERG a-wave amplitude at 30 cd.s/m2 flash intensity showed a progressive decrease to 36 ± 7.5 % (n = 6, p < 0.0005) as compared to control after 14 days. Similarly, the b-wave amplitude reduced to 36 ± 8 % (n = 6, p < 0.005). For control injection, there was an initial drop in ERG values on day 2 which completely recovered on days 4, 7, and 14 post injection. The c-wave amplitude was reduced to 50 ± 5 % (n = 5, p < 0.005) for Kir7.1 shRNA injection as compared to 86 ± 4.6 % (n = 4) for the control shRNA injected eye. The NaN3 electrical response with an average amplitude of 250 ± 9.6 µV (n = 8) was abolished in Kir7.1 shRNA injected mice.
Changes in subretinal space K+ concentration in light is restored to normal levels by RPE Kir7.1 channels. Knocking down Kcnj13 in the RPE using shRNA results in loss of Kir7.1 transcript and a severely reduced ERG in mice. Absence of NaN3 response further confirmed the loss of functional RPE cells. In both SVD and LCA, a loss of Kir7.1 channel function occurs, and as with the loss of Kcnj13 expression in our model, directly affects ERG outcome which can serve to non-invasively measure therapeutic results.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
This PDF is available to Subscribers Only