摘要：Despite efficient and specific in vitro knockdown, more reliable and convenient methods for in vivo knockdown of target genes remain to be developed particularly for retinal research. Using commercially available and chemically modified siRNA so-called Accell siRNA, we established a novel in vivo gene silencing approach in the rat retina. siRNA designed for knockdown of the house keeping gene Gapdh or four retinal cell type-specific genes (Nefl, Pvalb, Rho and Opn1sw) was injected into the vitreous body, and their retinal mRNA levels were quantified using real-time PCR. Intravitreal injection of siRNA for Gapdh resulted in approximately 40–70% reduction in its retinal mRNA levels, which lasted throughout a 9-day study period. Furthermore, all the selected retinal specific genes were efficiently down-regulated by 60–90% following intravitreal injection, suggesting injected siRNA penetrated into major retinal cell types. These findings were consistent with uniform distribution of a fluorescence-labeled siRNA injected into the vitreous body. Interestingly, gene silencing of Grin1, a core subunit of NMDA receptor, was accompanied by significant prevention from NMDA-induced retinal ganglion cell death. Thus, we provide single intravitreal injection of Accell siRNA as a versatile technique for robust and sustainable in vivo retinal gene silencing to characterize their biological functions under physiological and pathophysiological conditions.
其他摘要：Abstract Despite efficient and specific in vitro knockdown, more reliable and convenient methods for in vivo knockdown of target genes remain to be developed particularly for retinal research. Using commercially available and chemically modified siRNA so-called Accell siRNA, we established a novel in vivo gene silencing approach in the rat retina. siRNA designed for knockdown of the house keeping gene Gapdh or four retinal cell type-specific genes ( Nefl , Pvalb , Rho and Opn1sw ) was injected into the vitreous body, and their retinal mRNA levels were quantified using real-time PCR. Intravitreal injection of siRNA for Gapdh resulted in approximately 40–70% reduction in its retinal mRNA levels, which lasted throughout a 9-day study period. Furthermore, all the selected retinal specific genes were efficiently down-regulated by 60–90% following intravitreal injection, suggesting injected siRNA penetrated into major retinal cell types. These findings were consistent with uniform distribution of a fluorescence-labeled siRNA injected into the vitreous body. Interestingly, gene silencing of Grin1 , a core subunit of NMDA receptor, was accompanied by significant prevention from NMDA-induced retinal ganglion cell death. Thus, we provide single intravitreal injection of Accell siRNA as a versatile technique for robust and sustainable in vivo retinal gene silencing to characterize their biological functions under physiological and pathophysiological conditions.
