Identification of Allele-Specific RNAi Effectors Targeting Genetic Forms of Parkinson's Disease
Figure 2
Screening of A30P-targeting shRNAs against the heterozygous eGFP-tagged wild-type and mCherry-tagged A30P mutant α-synuclein expression plasmid.
A) eGFP-tagged wild-type α-synuclein and mCherry tagged A30P mutant α-synuclein were expressed in anti-parallel directions to one another in the Het A30P plasmid. B) Expression of the Het A30P plasmid resulted in robust eGFP fluorescence and robust mCherry fluorescence at 48 hrs post-transfection in HEK-293 cells. C+E) Representative merged fluorescent images of HEK-293 cells co-transfected with the Het-A30P plasmid and indicated single (C) or double (E) mismatch shRNA construct at 48 hrs post-transfection. D+F) Quantification of wild-type α-synuclein eGFP (green bars) or A30P mutant α-synuclein mCherry (red bars) fluorescence at 48 hrs post-transfection following co-transfection of single (D) or double (F) mismatch shRNAs targeting the A30P α-synuclein mutant with the Het-A30P plasmid. Values represent mean ratios of normalized fluorescence +/− S.D. from n = 6. Values are normalized to respective fluorescence in cells transfected with non-specific shRNA. * = P<0.05 relative to respective normalising control.