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A. Kiang, G.J. Farrar, P.F. Kenna, P. Humphries; Use of RNA Interference to Knockdown Expression of Mouse Rhodopsin in Cos-7 Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2335.
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Purpose:To investigate the efficacy of siRNAs targetting different regions of the mouse rhodopsin cDNA sequence in suppressing expression of mouse rhodopsin mRNA in Cos-7 cells. Methods:Five 23 bp sequences (AA(N19)TN) within mouse rhodopsin cDNA were selected. The sense and the antisense sequences separated by a 9 bp spacer sequence for each of the 5 chosen sites were separately cloned directly downstream of the human polymerase-III H1-RNA promoter within pBluescript SK(-) to give pHIMR1, pHIMR2, pHIMR3, pHIMR4 and pHIMR5. In addition a synthetic siRNA, siMR3, corresponding to siRNA sequences within pHIMR3, was synthesised commercially (Xeragon Inc). Cos-7 cells were transfected with a mouse rhodopsin cDNA clone and stable lines including cosMR3.1 isolated by selection with G418. Separate transfections of cosMR3.1 cells were carried out with the 5 plasmids pHIMR1-pHIMR5,a control plasmid pHICOL1A1 and with siMR3 and a control siRNA, siLZ1 using lipofectamine 2000 or oligofectin as described by the manufacturer (Invitrogen). Transfection efficiency was estimated by lacZ staining of cells transfected with pCMVbeta (BD Biosciences). 24h post transfection total RNA was extracted from the cells using Trizol (Gibco-BRL) and subjected to real time RT-PCR analysis using a LightCycler (Roche) Results:Mouse rhodopsin mRNA levels from cosMR3.1 cells transfected with plasmids pHIMR1-pHIMR5 showed a decrease of between 25% and 60% compared to cells transfected with pHICOL1A1. The estimated transfection efficiency was 60%. Plasmid pMR3 and siMR3 gave similar levels of suppression (60%). Conclusions:Taking transfection efficiency into consideration, the results suggest that plasmid pH1MR3 and siMR3 may suppress mouse rhodopsin mRNA with up to 100% efficiency in cosMR3.1 cells. Plasmids pHIMR1, pHIMR2, pHIMR4 and pHIMR5 showed lesser degrees of suppression indicating that within a given mRNA some regions can be targetted more eficiently than others by siRNA.
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