Translation of Is Specifically mRNA Injected into Xenopus Inhibited by Antisense RNA Oocytes

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 trans1094 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 effect maximal inhibition. Further, to address the question of whether antisense RNA can inhibit genes transcribed in the cell nucleus, we injected antisense CAT RNA into the oocyte cytoplasm and then tested the expression of a plasmid DNA coding for CAT injected into the oocyte nucleus. Our data complement and support the findings of Melton who has independently obtained similar results using Xenopus globin mRNA (13), and the results of Preiss et al. (16) who have shown that antisense RNA complementary to Kriipple mRNA can phenocopy a KrOpple mutation in Drosophila. MATERIALS AND METHODS Enzymes and Chemicals SP6 polymerase, radioactive nucleotides, and [~4C]chlorarnphenicol were obtained from New England Nuclear (Boston, MA); RNase inhibitor, from ~Abbreviations used in this paper. CAT, chloramphenicol acetyl transferase; TK, thymidine kinase. THE JOURNAL OF CELL BIOLOGY • VOLUME 101 SEPTEMBER 1985 1094 1099 © The Rockefeller University Press . 0021-9525/85/09/1094/06 $1.00 Promega Biotec (Madison, Wl); RNase-free DNase I and guanylyl transferase, from Bethesda Research Lab. (Bethesda, MD), and, nucleotides and acetyl CoA, from P L Biochemicals Inc. (Milwaukee, WI).