RNA interference rescue by bacterial artificial chromosome transgenesis in mammalian tissue culture cells

Abstract

RNA interference (RNAi) is a widely used method for analysis of gene function in tissue culture cells. However, to date there has been no reliable method for testing the specificity of any particular RNAi experiment. The ideal experiment is to rescue the phenotype by expression of the target gene in a form refractory to RNAi. The transgene should be expressed at physiological levels and with its different splice variants. Here, we demonstrate that expression of murine bacterial artificial chromosomes in human cells provides a reliable method to create RNAi-resistant transgenes. This strategy should be applicable to all eukaryotes and should therefore be a standard technology for confirming the specificity of RNAi. We show that this technique can be extended to allow the creation of tagged transgenes, expressed at physiological levels, for the further study of gene function.off-target effects ͉ loss-of-function experiment ͉ interferon response ͉ gene knockdown

Phenotypic characterization of missense polymerase-δ mutations using an inducible protein-replacement system

“…The latter approaches readily generate targeted genomic deletions, which cause full gene inactivation by non-homologous endjoining rather than a knockdown achieved by shRNA, but the introduction of specific mutations via homologous recombination is substantially less efficient, and requires expensive and timeconsuming screening for correct clones. Moreover, the system is not reversible, and rescue experiments require transfection of the cells with plasmid or bacmid vectors followed by clonal selection 12,13 , which could introduce artifacts. Our system controls for off-target effects of the shRNA by including a control that expresses the tagged WT variant in the presence of the shRNA.…”

“…An important shortcoming of RNAi technology are off-target effects 11 ; however, specificity can be demonstrated by rescue of the observed phenotype through expression of an RNAi-resistant mRNA 12,13 . Originally designed as a control experiment to check the specificity of RNAis, this approach has been extended to deploy RNAi-resistant mRNAs to express protein variants.…”

Tandem affinity purification of functional TAP-tagged proteins from human cells

“…In mammalian cells, the lack of efficient homologous recombination makes the functionality test difficult. Kittler et al 16 demonstrated that expression of murine bacterial artificial chromosomes (BAC) in human cells provides a reliable method to create RNA interference (RNAi)-resistant tagged transgenes. In such cells, the endogenous human gene can be knocked down by RNAi, while the corresponding murine gene expressed from the integrated BAC resists the RNAi treatment.…”

Short-hairpin RNA-mediated stable silencing of Grb2 impairs cell growth and DNA synthesis

“…Overexpression of XGrb2 SiL did not result in acute upregulation of endogenous Grb2 mRNA due to transfection artifacts, demonstrating that endogenous Grb2 silencing can be rescued at the protein level. Our laboratory has previously shown that Grb2, as part of a Shc-Grb2-SOS complex is essential for the signaling of the phospholipase D2 (PLD2) isoform through the SOS-RAS-RAF-MAPKK-MAPK signaling cascade and eventual effect on DNA synthesis and cell growth [12,13]. Efficient signaling requires association of the lipase with Grb2 through an identified residue, (as the Src homology 'SH' region-2 binds to phosphorylated tyrosine residues).…”