Facilitated in vivo synthesis of ribonucleic acid and protein via T7 RNA polymerase.
Ribozyme and small interfering RNA (siRNA) now are widely used to suppress target genes bearing homologous sequences. In this study, commonly used cell lines (e.g., HEK, HeLa, H1299) were stably transfected with gene encoding T7 RNA polymerase. The cytoplasm-restricted transcription activity of T7 RNA polymerase confers a continuous and robust transcription from T7 promoter-containing oligonucleotide (ODN) template for siRNA or ribozyme and leads to 70 to 80% inhibition of the tested target genes. ODN template offers the advantages of being more stable and economical than synthetic or in vitro-transcribed siRNA or ribozyme. Compared with the use of siRNA/ribozyme-expressing plasmids, our system does not require procedures with preparations of recombinant plasmids and enrichment of transfected cells and can be applied to synthesize protein in which different levels of translation could be modulated via variations in the presence of polyA tail or internal ribosome entry site (IRES) in the T7-transcribed RNAs. The results of our current study provide a rapid and efficient system for the assay of in vivo synthesis and expression of RNAs and proteins.
Wang
Tong-Hong
TH
Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan 11217, Republic of China.
Yu
Szu-Hsien
SH
Au
Lo-Chun
LC
D002460:Cell Line; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D009841:Oligonucleotides / Q000235:genetics; D014176:Protein Biosynthesis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000096:biosynthesis*; D016337:RNA, Catalytic / Q000378:metabolism; D034741:RNA, Small Interfering; D038621:Regulatory Sequences, Ribonucleic Acid / Q000235:genetics; D052243:Resistin / Q000378:metabolism; D013698:Templates, Genetic; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism*
D009841:Oligonucleotides; D019253:Proto-Oncogene Proteins c-bcl-2; D016337:RNA, Catalytic; D034741:RNA, Small Interfering; D038621:Regulatory Sequences, Ribonucleic Acid; D052243:Resistin; D016159:Tumor Suppressor Protein p53; D014764:Viral Proteins; D012313:RNA; D008156:Luciferases; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases