Constitutive and Conditional Suppression of Exogenous and Endogenous Genes by Anti-Sense RNA

Abstract

Plasmid DNA directing transcription of the noncoding (anti-sense) DNA strand can specifically inhibit the expression of several test genes as well as normal, endogenous genes. The anti-sense plasmid constructions can be introduced into eukaryotic cells by transfection or microinjection and function in both transient and stable transformation assays. Anti-sense transcripts complementary to as little as 52 bases of 5' untranslated target gene mRNA specifically suppress gene activity as well as, or more efficiently than, anti-sense transcripts directed against the protein coding domain alone. Conditional anti-sense inhibition is accomplished with the use of hormone-inducible promoter sequences. Suppression of endogenous actin gene activity by anti-sense RNA is detected as a decrease in growth rate and as a reduction in the number of actin microfilament cables. These observations suggest that anti-sense RNA may be generally useful for suppressing the expression of specific genes in vivo and may be a potential molecular alternative to classical genetic analysis.

Transcriptional control of the endogenous MYC protooncogene by antisense RNA.

“…To investigate the biological functions associated with a gene sequence, a valuable genetic approach is to introduce cloned DNA encoding antisense RNA into cells to specifically eliminate the gene product of interest or to block its function (1)(2)(3)(4). In the present work we examine the effects ofantisense MYC transcripts on the constitutive expression of the MYC gene and show that a human antisense MYC gene stably introduced into the human promyeloleukemia cell line HL-60 can inhibit not only MYC protein synthesis but also transcription of the endogenous MYC gene.…”

Translation of mRNA injected into Xenopus oocytes is specifically inhibited by antisense RNA.

“…Hybridization between such an antisense RNA and the normal message may prevent translation or processing. It was previously shown (7,8) that the expression of exogenous genes as well as endogenous cellular genes can be dramatically and specifically inhibited when DNA molecules that express antisense RNA have been introduced into tissue culture cells.Injection of plasmid DNA directing the production of antisense RNA into Xenopus fertilized eggs is possible, however a high concentration of injected DNA is toxic to embryos (4, 19), and the available expression plasmids may not produce large enough amounts of RNA quickly enough to inhibit genes turned on in the rapidly dividing embryo. In contrast, injected RNA is not as toxic and, at least in the case ofglobin RNA, is very stable in embryos (5).…”

Breeding for disease resistance ‐ the prospective role of genetic manipulation

“…Hybridisation of the antisense RNA to the mRNA in the cytoplasm and possibly also in the nucleus (Izant and Weintraub, 1985;Kim and Wold, 1985) inhibits its translation. The process is highly specific and tazra-acting but is dependent on the relative concentrations of mRNA and antisense RNA for the degree of inhibition.…”

“…The process is highly specific and tazra-acting but is dependent on the relative concentrations of mRNA and antisense RNA for the degree of inhibition. With a single antisense species 80 to 90% inhibition has been achieved (Izant and Weintraub, 1985;Green et al, 1986): it seems likely that the use of two or more antisense RNAs would give higher levels of inhibition. The effectiveness of the process has been demonstrated in a number of applications including the inhibition of devlopmental genes (Rosenberg et al, 1985;Crowley et al, 1985), viral genes (Izant and Weintraub, 1984) and of Rous sarcoma infection in vitro (Stephenson and Zamecnik, 1978;Zamecnik and Stephenson, 1978).…”