Expressing functional siRNAs in mammalian cells using convergent transcription.
BACKGROUND
The use of small interfering RNAs (siRNAs) as genetic inhibitors of gene expression has been shown to be an effective way of studying gene function in mammalian cells. Recently, different DNA vectors for expression of small hairpin RNAs (shRNAs) or co-expression of sense and antisense RNAs have been developed that direct siRNA-mediated gene silencing. One expression cassette design that has been used to express long sense and antisense RNAs in non-mammalian cell types is symmetric transcription using convergent promoters. However, convergent transcription as a way to generate functional siRNAs in mammalian cells has not been reported. This vector design permits the generation of expression constructs containing no repeat sequences, but capable of inducing RNA interference (RNAi)-mediated gene silencing.


RESULTS
With the aim of simplifying the construction of RNAi expression vectors, we report on the production and application of a novel convergent promoter cassette capable of expressing sense and antisense RNAs, that form double-stranded RNA, and mediate gene silencing in mammalian cells. We use this cassette to inhibit the expression of both the EGFP transgene and the endogenous TP53 gene. The gene silencing effect is Dicer-dependent and the level of gene inactivation achieved is comparable to that produced with synthetic siRNA. Furthermore, this expression system can be used for both short and long-term control of specific gene expression in mammalian cells.


CONCLUSIONS
The experiments performed in this study demonstrate that convergent transcription can be used in mammalian cells to invoke gene-specific silencing via RNAi. This method provides an alternative to expression of shRNAs and co-expression of sense and antisense RNAs from independent cassettes or a divergent promoter. The main advantage of the present vector design is the potential to produce a functional siRNA expression cassette with no repeat sequences. Furthermore, the cassette design reported is ideal for both routine use in controlling specific gene expression and construction of randomised RNAi expression libraries for use in unbiased forward genetic selections.
Tran
Nham
N
Johnson and Johnson Research Pty Ltd, 1 Central Ave, Australian Technology Park, Eveleigh, NSW 1430, Australia. nham@nucleics.com
Cairns
Murray J
MJ
Dawes
Ian W
IW
Arndt
Greg M
GM
D002460:Cell Line; D045744:Cell Line, Tumor; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009841:Oligonucleotides / Q000235:genetics; D016376:Oligonucleotides, Antisense / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D012342:RNA, Small Nuclear / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics*; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
D008164:Luminescent Proteins; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D034741:RNA, Small Interfering; D012342:RNA, Small Nuclear; D016159:Tumor Suppressor Protein p53; C405486:U6 small nuclear RNA; D049452:Green Fluorescent Proteins