Summary
An expression vector was constructed containing the entire bovine papilloma virus (BPV-1) genome and part of the a-actin gene of Xenopus laevis cloned in the antisense orientation into the neomycin resistance gene under the control of the herpes simplex virus (HSV) thymidine kinase (TK) promoter. When this vector is microinjected into X. laevis embryos it replicates extrachromosomally, at least up to the tadpole stage, and a fusion RNA is synthesized after the mid blastula transition (MBT). The expression of the antisense gene results in a morphological abnormality of somites demonstrating that antisense RNA generated by an episomal replicating expression vector can inhibit the expression of a selected gene during early embryogenesis of X. laevis.
Similar content being viewed by others
References
Akam M, Dawson I, Tear G (1988) Homeotic genes and the control of segment diversity. Development 104:123–133
Bass BL, Weintraub H (1987) A developmentally regulated activity that unwinds RNA duplexes. Cell 48:607–613
Budd PS, Jackson IJ (1991) What do the regulators regulate? First glimpses downstream. Trends Genet 7:74–76
Crowley TE, Nellen W, Gomer RH, Firtel RA (1985) Phenocopy of discoidin I-minus mutants by antisense transformation in Dictyostelium. Cell 43:633–641
Feinberg AP, Vogelstein B (1983) A technique for radiolabeling DNA restriction endonuclease fragments of high specific activity. Anal Biochem 132:6–13
Gehring WJ, Müller M, Affolter M, Percival-Smith A, Billeter M, Qian YQ, Otting G, Wüthrich K (1990) The structure of the homeodomain and its functional implications. Trends Genet 6: 323–329
Giebelhaus DH, Eib DW, Moon RT (1988) fnAntisense RNA inhibits expression of membrane skeleton protein 4.1 during embryonic development of Xenopus. Cell 53:601–615
Gurdon JB, Wakefield L (1986) Microinjection of amphibian oocytes and eggs for analysis of transcription. In: Graessmann JE, Loyter A (eds) Cells Microinjection and organelle transplantation techniques. Academic Press, New York, pp 271–299
Holt JT, Venkat-Gopal T, Moulton AD, Nienhus AW (1986) Inducible production of c-fos antisense RNA inhibits 3T3 proliferation. Proc Natl Acad Sci USA 83:4794–4798
Izant JG, Weintraub H (1984) Inhibition of thymidine kinase gene expression by antisense RNA: a molecular approach to genetic analysis. Cell 36:1007–1015
Kielbowna L (1981) The formation of somites and early myotomal myogenesis in Xenopus laevis, Bombina variegata and Pelobates fuscus. J Embryol Exp Morphol 64:295–304
Kim SK, Wold BJ (1985) Stable reduction of thymidine kinase activity in cells expressing high levels of antisense RNA. Cell 42:129–138
Kloc M, Miller M, Carrasco AE, Eastman E, Etkin L (1989) The maternal store of the xlgv7 mRNA in full-grown oocytes is not required for normal development in Xenopus. Development 107:899–907
Masho R (1990) Close correlation between the first cleavage plane and the body axis in early Xenopus embryos. Dev Growth Differ 32:57–64
Matthias PD, Bernard HU, Scott A, Brady G, Hashimoto-Gotoh T, Schütz G (1983) A bovine papilloma virus vector with a dominant resistance marker replicates extrachromosomally in mouse and E. coli eels. EMBO J 2:1487–1492
Melton DA (1985) Injected antisense RNAs specifically block messenger RNA translation in vivo. Proc Natl Acad Sci USA 82:144–148
Minshull J, Blow JJ, Hunt T (1989) Translation of cyclin mRNA is necessary for extracts of activated Xenopus eggs to enter mitosis. Cell 56:947–956
Nieuwkoop P, Faber J (1975) Normal table of Xenopus laevis. North Holland, Amsterdam
Rebagliati MR, Melton DA (1987) Antisense RNA injections in fertilized frog eggs reveal an RNA duplex unwinding activity. Cell 48:599–605
Rosenberg UB, Press A, Seifert E, Jäckle H, Knipple DC (1985) Production of phenocopies by Krüppel antinsense RNA injection into Drosophila embryos. Nature 313:703–706
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. Cold Spring Harbour Lab Press
Sanger F, Nickler S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Schmid M, Steinbeisser H, Trendelenburg MF, Lipps HJ (1990) A bovine papillomavirus type-1 (BPV-1) containing plasmid replicates extrachromosomally in Xenopus embryos. Nucl Acids Res 18:2196
Steinbeisser H, Hofmann A, Stutz F, Trendelenburg MF (1988) Different regulatory elements are required for cell-type and stage-specific expression of the Xenopus laevis skeletal muscle actin gene upon injection in Xenopus laevis oocytes and embryos. Nucl Acids Res 16:3223–3238
Stutz F, Spohr G (1986) Isolation and characterization of sarcomeric actin genes expressed in Xenopus laevis embryos. J Mol Biol 187:349–361
Weintraub H, Cheng PF, Conrad K (1986) Expression of transfected DNA depends on DNA topology. Cell 46:115–122
Wormington WK (1986) Stable repression of ribosomal protein L1 synthesis in Xenopus oocytes by microinjection of antisense RNA. Proc Natl Acad Sci USA 83:8639–8643
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Schmid, M., Steinbeisser, H., Epperlein, HH. et al. An expression vector inhibits gene expression in Xenopus embryos by antisense RNA. Roux's Arch Dev Biol 201, 340–345 (1992). https://doi.org/10.1007/BF00365121
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00365121