TRANSIENT EXPRESSION ASSAY FOR ANTISENSE RNAS USING EPISOMAL REPLICATION OF PLASMIDS - EFFECTIVE REDUCTION OF RETINOBLASTOMA GENE (RB-1) PRODUCT BY ITS ANTISENSE RNA COMPLEMENTARY TO 3'-UNTRANSLATED REGION
M. Kobayashi et al., TRANSIENT EXPRESSION ASSAY FOR ANTISENSE RNAS USING EPISOMAL REPLICATION OF PLASMIDS - EFFECTIVE REDUCTION OF RETINOBLASTOMA GENE (RB-1) PRODUCT BY ITS ANTISENSE RNA COMPLEMENTARY TO 3'-UNTRANSLATED REGION, Antisense research and development, 5(2), 1995, pp. 141-148
Citations number
26
Categorie Soggetti
Medicine, Research & Experimental","Biothechnology & Applied Migrobiology
We have developed a transient expression assay for selection of effect
ive antisense RNAs using episomal replication of plasmids in COS-7 cel
ls, an African green monkey kidney-derived cell line expressing SV40 l
arge T antigen. The transient expression assay was enabled by a liposo
me-Mediated DNA transfection method, by which about 70% of the cells w
ere reproducibly transfected with exogenous DNAs. Plasmids expressing
antisense RNAs for the retinoblastoma gene (Rb-1) mRNA and harboring S
V40 ori were constructed and introduced into COS-7 cells to examine th
eir inhibitory effect on the accumulation of endogenous Rb protein (pR
b). Only the antisense RNA complementary to the 3'-untranslated region
(UTR) in Rb-1 mRNA was expressed stably at high levels for 3 days aft
er the transfection. This antisense RNA reduced by 73% the content of
endogenous pRb 70 h after transfection. A similar inhibition was detec
ted in mouse mammary carcinoma cells (FM3A) that were stably transfect
ed with the antisense RNA expressing vector directed to 3'UTR. In cont
rast, no obvious change in pRb was observed with antisense RNAs comple
mentary to the coding region of Rb-1 mRNA. The cellular content of the
se antisense RNAs was lowered by degradation; thus these RNAs did not
affect the levels of pRb in COS-7 and FM3A cells. These results, taken
together, suggest that the expression levels and the stability of ant
isense RNAs are involved in their repressive activity, and our transie
nt expression assay provides a rapid and easy system for evaluation of
ectopic antisense RNA activity in COS-7 cells.