381. Methods for Optimizing Lentiviral Vector-Mediated Gene Suppression by RNA Interference

Abstract

Short interfering (si)RNA methods have the potential for specific suppression of target genes, but finding the optimal siRNA target sequence and evaluating the persistence of gene knockdown are challenges which hinder progress. We have developed a strategy for vector-based screening of multiple siRNA sequences against target genes in mammalian cells. The steps are as follows: 1) creation of a fusion construct of the gene of interest (GOI) and a fluorescent reporter such as GFP at either the N-or C-terminus, 2) stable expression of the GOI-GFP fusion in a mammalian cell line, 3) transfection of vector plasmids encoding templates for short-hairpin (sh)RNAs, under the transcriptional control of a human RNA polymerase III (H1) promoter, targeted against several GOI sequences in the stable cell line, 4) quantification of the GOI knockdown by flow cytometric analysis for diminished GFP fluorescence. Quantification of expression by this method permits not only the selection of the most suppressive sequences but also those capable of resulting in intermediate levels of suppresion and hence intermediate phenotypic outcomes. In a modification of step 3, the shRNA expression cassettes are subcloned into a lentiviral vector system permitting transduction of stably modified cells thereby enabling the evaluation of persistence of the suppressive effect after multiple subcultures.