Inhibition of thymidine kinase gene expression by anti-sense RNA: A molecular approach to genetic analysis

Izant, Jonathan G.; Weintraub, Harold

doi:10.1016/0092-8674(84)90050-3

Cited by 527 publications

(225 citation statements)

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“…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).…”

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confidence: 99%

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

Harland¹,

Weintraub²

1985

The Journal of Cell Biology

192

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The bacteriophage SP6 promoter and RNA polymerase were used to synthesize sense and antisense RNAs coding for the enzymes thymidine kinase (TK) and chloramphenicol acetyl transferase (CAT). Injection of antisense CAT RNA into frog oocytes inhibited expression of sense CAT mRNA. Similarly, antisense TK RNA inhibited expression of sense TK mRNA. Antisense RNAs were stable in oocytes and had no detectable effect on either the expression of endogenous proteins or on the expression of nonhomologous RNA transcripts. CAT activity expressed from a plasmid transcribed in the oocyte nucleus was also inhibited by antisense RNA injected into the oocyte cytoplasm. The data suggest that antisense RNA will be useful in identifying the function of specific mRNA sequences during early development of the frog.We are trying to identify and characterize genes that participate in normal embryonic development in the frog Xenopus laevis. An informative method of studying such genes would be to inhibit their expression in vivo to directly identify and assess the biological functions to which the genes contribute. While Xenopus embryos are a favorable system for experimental manipulations and biochemical analyses, the organism is not readily available to classical genetic analysis. One other way that genes may be inhibited is by the introduction of RNA complementary in sequence to the normal message (7). 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).Recently it has become possible to synthesize large amounts of a specific RNA from a plasmid containing a segment of cloned DNA downstream from the SP6 promoter (3,14). Using this technology, we have synthesized sense mRNAs coding for the bacterial enzyme chloramphenicol acetyl trans-1094 ferase (CAT) ~ and herpes simplex virus thymidine kinase (TK), as well as their antisense counterparts. We have first injected antisense, then sense mRNA into oocytes of Xenopus, and then extracted proteins for enzyme assay. Using these methods and quantitative measurements of sense and antisense RNA half-lives, we have determined (a) the extent to which expression of these sense mRNAs is inhibited at different concentrations of antisense RNA, and (b) whether an antisense RNA covering the full length of the protein-coding region of mRNA is necessary to eff...

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mentioning

confidence: 99%

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

Harland¹,

Weintraub²

1985

The Journal of Cell Biology

192

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“…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.…”

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confidence: 99%

Transcriptional control of the endogenous MYC protooncogene by antisense RNA.

Yokoyama

Imamoto

1987

Proc. Natl. Acad. Sci. U.S.A.

129

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DNAs (20 ,ug) by the protoplast fusion method (10). Two days later, selection for the ability to grow in Dulbecco's modified Eagle's medium (DMEM) containing mycophenolic acid (25 ,gg/ml), aminopterin, xanthine, hypoxanthine, and thymidine was performed as described by Mulligan and Berg (11). Transformants resistant to a high dose of mycophenolic acid (110 Ag/ml) were obtained by culturing cells in DMEM plus mycophenolic acid over a period of 6 months during which the mycophenolic acid concentration was increased at 2-week intervals. Secondary and tertiary transformants were obtained by retransfecting a primary and a secondary transformant, respectively, with plasmid DNA (10 ,ug/ml) followed by culturing in DMEM containing mycophenolic acid (110 ,g/ml). Control antisense and sense clones (pSV2gpt+, pSVgptC5-8, pSVH-2Kb, and pSVa-globin) were obtained by the cotransfection with pSV2gpt+. The same amplification protocol and the repeated transfection were used as described above.RNA Isolation and Analysis. Total RNA, poly(A)+ mRNA, and cytoplasmic and nuclear RNAs were prepared by the standard protocols (12, 13). RNA gel blot transfer and hybridization were carried out as described by Maniatis et al.(13). [a-32P]dCTP-labeled single-stranded probes were prepared from restriction fragments containing f,-actin cDNA (14) or exons 2 and 3 of MYC cDNA by exonuclease III digestion, reverse transcriptase treatment (15), and strand separation (13 7363The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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“…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).…”

Section: Genes For Insertionmentioning

confidence: 99%