A novel bidirectional expression system for simultaneous expression of both the protein-coding genes and short hairpin RNAs in mammalian cells.
RNA interference (RNAi) is an extremely powerful and widely used gene silencing approach for reverse functional genomics and molecular therapeutics. In mammals, the conserved poly(ADP-ribose) polymerase 2 (PARP-2)/RNase P bidirectional control promoter simultaneously expresses both the PARP-2 protein and RNase P RNA by RNA polymerase II- and III-dependent mechanisms, respectively. To explore this unique bidirectional control system in RNAi-mediated gene silencing strategy, we have constructed two novel bidirectional expression vectors, pbiHsH1 and pbiMmH1, which contained the PARP-2/RNase P bidirectional control promoters from human and mouse, for simultaneous expression of both the protein-coding genes and short hairpin RNAs. Analyses of the dual transcriptional activities indicated that these two bidirectional expression vectors could not only express enhanced green fluorescent protein as a functional reporter but also simultaneously transcribe shLuc for inhibiting the firefly luciferase expression. In addition, to extend its utility for the establishment of inherited stable clones, we have also reconstructed this bidirectional expression system with the blasticidin S deaminase gene, an effective dominant drug resistance selectable marker, and examined both the selection and inhibition efficiencies in drug resistance and gene expression. Moreover, we have further demonstrated that this bidirectional expression system could efficiently co-regulate the functionally important genes, such as overexpression of tumor suppressor protein p53 and inhibition of anti-apoptotic protein Bcl-2 at the same time. In summary, the bidirectional expression vectors, pbiHsH1 and pbiMmH1, should provide a simple, convenient, and efficient novel tool for manipulating the gene function in mammalian cells.
Hung
Chuan-Fu
CF
Department of Biochemistry and Molecular Biology, National Cheng Kung University Medical College, No. 1, University Road, Tainan 701, Taiwan, ROC.
Cheng
Tsung-Lin
TL
Wu
Ren-Huang
RH
Teng
Chiao-Fang
CF
Chang
Wen-Tsan
WT
D000818:Animals; D002478:Cells, Cultured; D006224:Cricetinae; D005786:Gene Expression Regulation / Q000235:genetics; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D007668:Kidney / Q000378:metabolism; D016366:Open Reading Frames / Q000235:genetics*; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*
D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C483041:PARP2 protein, human; D011065:Poly(ADP-ribose) Polymerases