RNA interference rescue by bacterial artificial chromosome transgenesis in mammalian tissue culture cells.
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.
Kittler
Ralf
R
Max Planck Institute for Molecular Cell Biology and Genetics, 01307 Dresden, Germany.
Pelletier
Laurence
L
Ma
Chunling
C
Poser
Ina
I
Fischer
Steffi
S
Hyman
Anthony A
AA
Buchholz
Frank
F
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D015870:Gene Expression; D050956:HSP40 Heat-Shock Proteins; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000235:genetics; D006801:Humans; D051379:Mice; D010641:Phenotype; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection
C485636:Dnaja3 protein, mouse; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D034741:RNA, Small Interfering