Inhibition of interleukin-1beta (IL-1beta) production in human cells by ribozymes against IL-1beta and IL-1beta converting enzyme (ICE).
We and others have shown previously that hairpin ribozyme genes, when stably expressed in cells, can reduce the steady-state levels of target mRNA and their cognate proteins. Despite this capability, ribozymes have not been as widely used in knockdown experiments as one might expect, probably because specific rules governing the selection of ribozymes that will have high activity have not been described. In this report, we show that parallel screening of less than 10 ribozyme expression constructs, with no advanced knowledge of cleavage activity or preselection, can efficiently identify knockdown ribozymes. This empirical selection study, which used interleukin-1beta (IL-1beta) and IL-1beta converting enzyme (ICE) as example targets, resulted in (1) the rapid identification of ribozymes that can reduce the production of IL-1beta in THP-1 cultures by 10-fold and (2) the consequent direct generation of stable knockdown cell lines. We conclude, based on these and similar studies, that parallel screening of ribozyme constructs could be used in high throughput gene functional analysis programs as a means of rapidly generating specific knockdown cell lines.
Leavitt
M C
MC
Immusol Incorporated, San Diego, CA 92121, USA.
Yu
G
G
Zhou
C
C
Barber
J R
JR
D020170:Caspase 1 / Q000378:metabolism; D061945:Caspase Inhibitors; D004797:Enzyme-Linked Immunosorbent Assay; D018628:Gene Dosage; D006801:Humans; D007375:Interleukin-1 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016337:RNA, Catalytic / Q000235:genetics / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D061945:Caspase Inhibitors; D007375:Interleukin-1; D016337:RNA, Catalytic; C418481:hairpin ribozyme; D020170:Caspase 1