A novel T7 system utilizing mRNA coding for T7 RNA polymerase.
The T7 system dose not require the relocation of a reporter gene to the nucleus for its gene expression in the cytoplasm, but relies on the co-localization of T7 RNA polymerase (T7 RNAP) enzyme and reporter gene DNA that is controlled by the T7 promoter. In the present study, we developed a new T7 system in that gene expression can occur at a higher level than those using conventional systems. Insertion of 5'- and 3'-untranslated regions (UTR) of beta-globin gene into a reporter gene enhanced the reporter gene expression, presumably due to the stability and efficient translation of the mRNA. Instead of the T7 RNAP protein used in conventional methods, moreover, transfection of cells with T7 RNAP mRNA, which has been modified by inserting beta-globin 5'- and 3'-UTR sequences as well as the cap and poly(A) tail structures, further enhanced the reporter gene expression. Thus, this novel T7 system using T7 RNAP mRNA may be powerful for the efficient gene expression of DNA exogenously provided in the cytoplasm.
Nakano
Risako
R
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, 565, Osaka, Japan.
Nakagawa
Tetsuhiko
T
Imazu
Susumu
S
Katayama
Kazufumi
K
Mizuguchi
Hiroyuki
H
Hayakawa
Takao
T
Tsutsumi
Yasuo
Y
Nakagawa
Shinsaku
S
Mayumi
Tadanori
T
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D000818:Animals; D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics*; D002460:Cell Line; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D015870:Gene Expression; D017930:Genes, Reporter; D005821:Genetic Techniques; D005914:Globins / Q000235:genetics; D008156:Luciferases / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection; D014764:Viral Proteins
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D012333:RNA, Messenger; D012367:RNA, Viral; D014764:Viral Proteins; D005914:Globins; D008156:Luciferases; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases