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C. Liu, T. Nguyen, Q. Cheng, M. Calvin, J. Bonanno; In vivo Knockdown of Sodium-Bicarbonate Cotransporter NBCe1 in Rabbit Corneal Endothelium by Lentiviral Gene Delivery. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3173. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate in vivo function of the sodium-bicarbonate cotransporter, NBCe1, in corneal endothelium.
An shRNA against NBCe1 was cloned into FIV-derived RNAi lentivector (pFIV-H1-copGFP) and assembled into the lenti-pseudoviral packaging system (pPACKF1). In the same vector, copepod green fluorescent protein (copGFP) was expressed as a reporter gene. Pseudoviral titer was determined according to the fraction of fluorescent cells by FACS analysis in primary cultured rabbit corneal endothelial cells. Up to 106 RNAi-NBCe1 pseudoviral particles in 30 µl PBS plus 8 µg/ml Polybrene were injected into the anterior chamber of one eye of a 7-month -old New Zealand white rabbit. The other eye was mock injected with 35 µl PBS plus 8 µg/ml Polybrene as control. CopGFP fluorescence was detected by a laser scanning confocal ophthalmoscope (HRA2). Central corneal thickness and IOP were measured by ultrasound Pachymeter and Tonopen, respectively. Brinzolamide 1%, a carbonic anhydrase inhibitor, was applied to stress the cornea. The rabbit was sacrificed and eyes were enucleated four weeks post pseudoviral injection. Fresh corneal endothelium was dissected from both eyes and analyzed for protein expression of NBCe1 and copGFP.
At least 1 x 106 pseudoviral particles were needed to detect copGFP by laser scanning confocal ophthalmoscopy. This dose also resulted in significant knockdown of NBCe1 by immunofluorescent analysis. However, anterior chamber inflammation was apparent at 24 hours and 2 weeks post injection. Addition of one drop of Brinzolamide caused 7.3 % corneal swelling in the control eye and 14.6 % in the NBCe1 knockdown eye. Following injection of lower dose of pseudovirus (2.5x105 particles/ ml), copGFP fluorescence was undetected by HRA2. Nevertheless two drops of Brinzolamide induced 5.3% corneal swelling in the control eye, and 13.1 % in the NBCe1 knockdown eye. At this dose, inflammation was undetected at 2 weeks post injection. Four weeks post injection, Western blot analysis revealed 74.5 % NBCe1 knockdown in the RNAi-NBCe1 pseudoviral injected eye compared to the control eye. Furthermore, copGFP was detectable by Western blot. Baseline central corneal thickness and IOP were unaffected.
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