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Joo Yong Lee, YU SUN KIM, CHANGMO HWANG; Channelrhodopsin-2 gene transduction to rat retina using sonoporation . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5896.
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© ARVO (1962-2015); The Authors (2016-present)
Ultrasound targeted microbubble destruction (UTMD) method has been used to deliver pcDNA3.1/hChR2(H134R)-mCherry plasmid into retinal pigment epithelium (RPE) in vitro and neurosensory retina in vivo settings. This study was performed to investigate the possibility of pcDNA transduction into RPE cell in vitro using UTMD in vivo ARPE-19 cells and neurosensory retina in vivo.
Different doses of pcDNA3.1/hChR2-mCherry plasmid were used under the different conditions of power, duty cycle, and application time using UTMD method in vitro and vivo. In vivo, pcDNA3.1/hChR2-mCherry was injected into the subretinal space of rat eyes. Transduction was assessed by fluorescence microscopy in vitro and both of fluorescence microscopy and confocal laser scanning microscopy in vivo. Tissue damage was also assessed by hematoxylineosin (H&E) staining.
Even though the transduction efficiency is very low, pcDNA3.1/hChR2-mCherry positive cells can be seen after UTMD. Also, the efficiency was different and could be improved depending on the different setting of UTMD method. In the transduction of pcDNA3.1/hChr2-mCherry into the neurosensory retina in vivo setting, mCherry positive cells also identified in the area of subretinal injection. No definite cell damages or structural changes were found in the posterior pole using H&E staining.
These methods might be used as an one of the way to transfer the target gene into the neurosensory retina in the eyes with retinal diseases.
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