title: Reconstitution of CKMT1 expression fails to rescue cells from mitochondrial membrane potential dissipation: implications for controlling RNAi experiments.
abstract: RNA interference (RNAi) is an essential method in molecular biology to reduce the expression of target genes and thereby determine their function. Since this tool is known to also have unspecific effects, control experiments are needed, chiefly among them the exclusion of off-target effects and the reconstitution of the genes' expression for the rescue of the cellular RNAi effects. We show here that the knock-down of the mitochondrial creatine kinase-1 (CKMT1) by RNA interference causes the dissipation of the mitochondrial membrane potential ΔΨm. This was accomplished with 11 different RNAi constructs designed to target 7 distinct exons as well as exon/intron junctions making unspecific off-target effects unlikely. However, all our attempts failed to rescue human cells from ΔΨm dissipation by the expression of CKMT1 alleles not targeted by RNAi. This included the transient and stable expression of the murine CKMT1 homologue, the expression of human codon usage-modified alleles, the transfection of a novel splice-isoform of CKMT1, and even the introduction of a human genomic clone for CKMT1 with codon usage changes. Our results indicate that while off-target effects of RNA interference can easily be addressed, the rescue of the knock-down phenotype is not necessarily achievable.
authors.affiliation: Division of Experimental Medicine, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 0NN, UK.
authors.forename: Christoph
authors.initials: C
authors.lastname: Datler
authors.affiliation: Division of Experimental Medicine, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 0NN, UK. Electronic address: s.grimm@imperial.ac.uk.
authors.forename: Stefan
authors.initials: S
authors.lastname: Grimm
mesh_terms: D017398:Alternative Splicing / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D003062:Codon / Q000235:genetics; D003402:Creatine Kinase / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D015894:Genome, Human / Q000235:genetics; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D053078:Membrane Potential, Mitochondrial; D051379:Mice; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D010641:Phenotype; D020033:Protein Isoforms / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: D003062:Codon; D050505:Mutant Proteins; D020033:Protein Isoforms; D034741:RNA, Small Interfering; C519581:CKMT1A protein, human; D003402:Creatine Kinase
keywords: Creatine kinase-1; Phenotype rescue; RNA interference