A novel class of temperature-sensitive mutants generated by RNAi-mediated knockdown.
Temperature-sensitive (ts) mutants are powerful tools with which to investigate gene function, but it has been difficult to generate ts mutants in mammalian cells. Recently, RNA interference (RNAi) has been widely used for loss of function analyses. In addition, in various organisms, hypothermic-temperature-sensitive RNAi has been reported. By using this characteristic of RNAi, we attempted to generate ts mutants in mammalian cells and were able to successfully generate ts mutants of cell cycle regulator cdc2 and ubiquitin-activating enzyme E1. We compared ts mutants previously isolated by mutagenesis with those generated by RNAi knockdown, and observed similar phenotypes. This method enabled us to generate ts mutants (KDts, knockdown temperature-sensitive mutants) of the genes of interest and will be utilized to facilitate understanding of the biological processes regulated by an essential gene in mammalian cells.
Inaki
Makoto
M
Genome and Drug Research Center, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
Utsugi
Takahiko
T
Onoda
Fumitoshi
F
Hanaoka
Fumio
F
Murakami
Yasufumi
Y
D000818:Animals; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors* / Q000235:genetics; D045744:Cell Line, Tumor; D005821:Genetic Techniques; D049452:Green Fluorescent Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D006358:Hot Temperature; D051379:Mice; D034622:RNA Interference; D012190:Retroviridae / Q000235:genetics; D013696:Temperature; D044764:Ubiquitin-Activating Enzymes / Q000037:antagonists & inhibitors* / Q000235:genetics
D049452:Green Fluorescent Proteins; D016203:CDC2 Protein Kinase; D044764:Ubiquitin-Activating Enzymes