Ribozyme expression systems.
We have succeeded in constructing an effective system for the expression of ribozymes under the control of a human tRNAVal promoter, which ensures a high level of production of ribozymes in vivo. The engineered tRNAVal-driven ribozymes, based on computer-predicted secondary structure, were relatively stable and were transported to the cytoplasm, where they could be colocalized with their target RNA. The activity of the exported ribozymes was significantly higher than that of ribozymes that remained in the nucleus. This chapter examines the methods for construction of the appropriate vector that can produce tRNA-driven ribozymes with high-level intracellular activity and for analysis of the constructs in cells.
Sano
Masayuki
M
Gene Function Research Center, National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan.
Taira
Kazunari
K
D001483:Base Sequence; D017931:DNA Primers; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic / Q000235:genetics; D016337:RNA, Catalytic / Q000235:genetics* / Q000378:metabolism*; D012366:RNA, Transfer, Val / Q000235:genetics*; D015183:Restriction Mapping; D013379:Substrate Specificity; D014158:Transcription, Genetic / Q000235:genetics*; D014162:Transfection
D017931:DNA Primers; D016337:RNA, Catalytic; D012366:RNA, Transfer, Val; D008156:Luciferases