hTERT knockdown in human embryonic kidney cells using double-stranded RNA.
The method of RNA interference (RNAi) is an easy means of knocking down a gene without having to generate knockout mutants, which may prove to be difficult and time consuming. RNAi is a naturally occurring process that involves targeting the mRNA of a gene by introducing RNAs that are complementary to the target mRNA. The foreign RNAs activate an endogenous enzyme, DICER, which degrades the target mRNA. There are many ways of eliciting the RNAi response in a cell. In this chapter, we describe the use of double-stranded RNA (dsRNA) to knockdown human telomerase reverse transcriptase (hTERT), the gene that codes for the catalytic subunit of the human telomerase enzyme. dsRNA can be used to generate the RNAi response in cells of embryonic origin, such as human embryonic kidney (HEK) cells. The RNAi effect is transient because the dsRNA eventually gets degraded in the cells, and it is useful to study the short-term effects of a gene knockdown.
Lai
Serene R
SR
Department of Biology, University of Alabama at Birmingham, Birmingham, AL, USA.
Andrews
Lucy G
LG
Tollefsbol
Trygve O
TO
D002460:Cell Line; D004587:Electrophoresis, Agar Gel; D006801:Humans; D008967:Molecular Biology / Q000379:methods*; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics / Q000494:pharmacology*; D019098:Telomerase / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
D012330:RNA, Double-Stranded; C509186:TERT protein, human; D019098:Telomerase