Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1992 Nov 1;89(21):10051–10055. doi: 10.1073/pnas.89.21.10051

Antisense c-myc effects on preimplantation mouse embryo development.

B C Paria 1, S K Dey 1, G K Andrews 1
PMCID: PMC50275  PMID: 1279668

Abstract

Antisense DNA inhibition of gene expression was explored as an approach toward elucidating mechanisms regulating development of preimplantation mammalian embryos. Specifically, a role for the c-myc protooncogene was examined. Detection of c-myc mRNA and immunoreactive nuclear c-myc protein in preimplantation mouse embryos at the eight-cell/morula and blastocyst stages suggested that this DNA-binding protein could be important during early embryo-genesis. The effects of c-myc oligodeoxyribonucleotides (oligos) on the in vitro development of two-cell mouse embryos were examined. Embryos cultured in medium containing an unmodified (phosphodiester) antisense c-myc oligo complementary to the translation initiation codon and spanning the first seven codons exhibited a dose-dependent arrest at the eight-cell/morula stage. At lower concentrations (7.5 microM) this inhibitory effect was specific to the antisense oligo and did not occur with the sense-strand complement or with duplexes of the antisense and sense oligos. However, at 4-fold higher concentrations of DNA (30 microM), all unmodified c-myc oligos were embryotoxic, causing embryos to arrest at the two-cell to four-cell stages. In contrast, almost all (98%) two-cell embryos cultured with a modified (chimeric phosphorothioate/phosphodiester) antisense c-myc oligo (7.5 microM) exhibited developmental arrest at the eight-cell/morula stage, whereas no developmental arrest occurred following incubation with high concentrations of the modified sense complement (30 microM). Culture of freshly recovered eight-cell embryos with antisense c-myc led to the absence of c-myc protein but no change in epidermal growth factor receptor in those embryos that developed a blastocoel. These effects on c-myc were specific for the antisense oligo. These results suggest that c-myc function becomes particularly critical for preimplantation mouse embryos at the eight-cell/morula stage of development and establish that antisense DNA can be successfully applied as an approach toward elucidating the roles of specific genes in preimplantation mammalian embryo development.

Full text

PDF
10051

Images in this article

10052Image
on p.10052
10053Image
on p.10053
10054Image
on p.10054

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andrews G. K., Huet-Hudson Y. M., Paria B. C., McMaster M. T., De S. K., Dey S. K. Metallothionein gene expression and metal regulation during preimplantation mouse embryo development (MT mRNA during early development). Dev Biol. 1991 May;145(1):13–27. doi: 10.1016/0012-1606(91)90209-l. [DOI] [PubMed] [Google Scholar]
  2. Ao A., Erickson R. P., Bevilacqua A., Karolyi J. Antisense inhibition of beta-glucuronidase expression in preimplantation mouse embryos: a comparison of transgenes and oligodeoxynucleotides. Antisense Res Dev. 1991 Spring;1(1):1–10. doi: 10.1089/ard.1991.1.1. [DOI] [PubMed] [Google Scholar]
  3. Bass B. L., Weintraub H. A developmentally regulated activity that unwinds RNA duplexes. Cell. 1987 Feb 27;48(4):607–613. doi: 10.1016/0092-8674(87)90239-x. [DOI] [PubMed] [Google Scholar]
  4. Bevilacqua A., Loch-Caruso R., Erickson R. P. Abnormal development and dye coupling produced by antisense RNA to gap junction protein in mouse preimplantation embryos. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5444–5448. doi: 10.1073/pnas.86.14.5444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bishop J. M. Cellular oncogenes and retroviruses. Annu Rev Biochem. 1983;52:301–354. doi: 10.1146/annurev.bi.52.070183.001505. [DOI] [PubMed] [Google Scholar]
  6. Blackwell T. K., Kretzner L., Blackwood E. M., Eisenman R. N., Weintraub H. Sequence-specific DNA binding by the c-Myc protein. Science. 1990 Nov 23;250(4984):1149–1151. doi: 10.1126/science.2251503. [DOI] [PubMed] [Google Scholar]
  7. Blackwood E. M., Eisenman R. N. Max: a helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc. Science. 1991 Mar 8;251(4998):1211–1217. doi: 10.1126/science.2006410. [DOI] [PubMed] [Google Scholar]
  8. Blanchard J. M., Piechaczyk M., Dani C., Chambard J. C., Franchi A., Pouyssegur J., Jeanteur P. c-myc gene is transcribed at high rate in G0-arrested fibroblasts and is post-transcriptionally regulated in response to growth factors. Nature. 1985 Oct 3;317(6036):443–445. doi: 10.1038/317443a0. [DOI] [PubMed] [Google Scholar]
  9. Dony C., Kessel M., Gruss P. Post-transcriptional control of myc and p53 expression during differentiation of the embryonal carcinoma cell line F9. Nature. 1985 Oct 17;317(6038):636–639. doi: 10.1038/317636a0. [DOI] [PubMed] [Google Scholar]
  10. Downs K. M., Martin G. R., Bishop J. M. Contrasting patterns of myc and N-myc expression during gastrulation of the mouse embryo. Genes Dev. 1989 Jun;3(6):860–869. doi: 10.1101/gad.3.6.860. [DOI] [PubMed] [Google Scholar]
  11. Goldman D. S., Kiessling A. A., Cooper G. M. Post-transcriptional processing suggests that c-mos functions as a maternal message in mouse eggs. Oncogene. 1988 Aug;3(2):159–162. [PubMed] [Google Scholar]
  12. Green P. J., Pines O., Inouye M. The role of antisense RNA in gene regulation. Annu Rev Biochem. 1986;55:569–597. doi: 10.1146/annurev.bi.55.070186.003033. [DOI] [PubMed] [Google Scholar]
  13. Heikkila R., Schwab G., Wickstrom E., Loke S. L., Pluznik D. H., Watt R., Neckers L. M. A c-myc antisense oligodeoxynucleotide inhibits entry into S phase but not progress from G0 to G1. 1987 Jul 30-Aug 5Nature. 328(6129):445–449. doi: 10.1038/328445a0. [DOI] [PubMed] [Google Scholar]
  14. Herzog N. K., Singh B., Elder J., Lipkin I., Trauger R. J., Millette C. F., Goldman D. S., Wolfes H., Cooper G. M., Arlinghaus R. B. Identification of the protein product of the c-mos proto-oncogene in mouse testes. Oncogene. 1988 Aug;3(2):225–229. [PubMed] [Google Scholar]
  15. Hirning U., Schmid P., Schulz W. A., Kozak L. P., Hameister H. In developing brown adipose tissue c-myc protooncogene expression is restricted to early differentiation stages. Cell Differ Dev. 1989 Sep;27(3):243–248. doi: 10.1016/0922-3371(89)90704-1. [DOI] [PubMed] [Google Scholar]
  16. Holt J. T., Redner R. L., Nienhuis A. W. An oligomer complementary to c-myc mRNA inhibits proliferation of HL-60 promyelocytic cells and induces differentiation. Mol Cell Biol. 1988 Feb;8(2):963–973. doi: 10.1128/mcb.8.2.963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Huet-Hudson Y. M., Andrews G. K., Dey S. K. Cell type-specific localization of c-myc protein in the mouse uterus: modulation by steroid hormones and analysis of the periimplantation period. Endocrinology. 1989 Sep;125(3):1683–1690. doi: 10.1210/endo-125-3-1683. [DOI] [PubMed] [Google Scholar]
  18. Huet-Hudson Y. M., Chakraborty C., De S. K., Suzuki Y., Andrews G. K., Dey S. K. Estrogen regulates the synthesis of epidermal growth factor in mouse uterine epithelial cells. Mol Endocrinol. 1990 Mar;4(3):510–523. doi: 10.1210/mend-4-3-510. [DOI] [PubMed] [Google Scholar]
  19. Kaczmarek L., Hyland J. K., Watt R., Rosenberg M., Baserga R. Microinjected c-myc as a competence factor. Science. 1985 Jun 14;228(4705):1313–1315. doi: 10.1126/science.4001943. [DOI] [PubMed] [Google Scholar]
  20. Kelly K., Cochran B. H., Stiles C. D., Leder P. Cell-specific regulation of the c-myc gene by lymphocyte mitogens and platelet-derived growth factor. Cell. 1983 Dec;35(3 Pt 2):603–610. doi: 10.1016/0092-8674(83)90092-2. [DOI] [PubMed] [Google Scholar]
  21. Kimelman D., Kirschner M. W. An antisense mRNA directs the covalent modification of the transcript encoding fibroblast growth factor in Xenopus oocytes. Cell. 1989 Nov 17;59(4):687–696. doi: 10.1016/0092-8674(89)90015-9. [DOI] [PubMed] [Google Scholar]
  22. Kingston R. E., Baldwin A. S., Jr, Sharp P. A. Regulation of heat shock protein 70 gene expression by c-myc. Nature. 1984 Nov 15;312(5991):280–282. doi: 10.1038/312280a0. [DOI] [PubMed] [Google Scholar]
  23. Mutter G. L., Wolgemuth D. J. Distinct developmental patterns of c-mos protooncogene expression in female and male mouse germ cells. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5301–5305. doi: 10.1073/pnas.84.15.5301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nau M. M., Brooks B. J., Battey J., Sausville E., Gazdar A. F., Kirsch I. R., McBride O. W., Bertness V., Hollis G. F., Minna J. D. L-myc, a new myc-related gene amplified and expressed in human small cell lung cancer. Nature. 1985 Nov 7;318(6041):69–73. doi: 10.1038/318069a0. [DOI] [PubMed] [Google Scholar]
  25. Nishikura K., Murray J. M. Antisense RNA of proto-oncogene c-fos blocks renewed growth of quiescent 3T3 cells. Mol Cell Biol. 1987 Feb;7(2):639–649. doi: 10.1128/mcb.7.2.639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nishikura K., Murray J. M. The mechanism of inactivation of the normal c-myc gene locus in human Burkitt lymphoma cells. Oncogene. 1988 May;2(5):493–498. [PubMed] [Google Scholar]
  27. O'Keefe S. J., Wolfes H., Kiessling A. A., Cooper G. M. Microinjection of antisense c-mos oligonucleotides prevents meiosis II in the maturing mouse egg. Proc Natl Acad Sci U S A. 1989 Sep;86(18):7038–7042. doi: 10.1073/pnas.86.18.7038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Paria B. C., Dey S. K. Preimplantation embryo development in vitro: cooperative interactions among embryos and role of growth factors. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4756–4760. doi: 10.1073/pnas.87.12.4756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Persson H., Hennighausen L., Taub R., DeGrado W., Leder P. Antibodies to human c-myc oncogene product: evidence of an evolutionarily conserved protein induced during cell proliferation. Science. 1984 Aug 17;225(4663):687–693. doi: 10.1126/science.6431612. [DOI] [PubMed] [Google Scholar]
  30. Persson H., Leder P. Nuclear localization and DNA binding properties of a protein expressed by human c-myc oncogene. Science. 1984 Aug 17;225(4663):718–721. doi: 10.1126/science.6463648. [DOI] [PubMed] [Google Scholar]
  31. Pfeifer-Ohlsson S., Rydnert J., Goustin A. S., Larsson E., Betsholtz C., Ohlsson R. Cell-type-specific pattern of myc protooncogene expression in developing human embryos. Proc Natl Acad Sci U S A. 1985 Aug;82(15):5050–5054. doi: 10.1073/pnas.82.15.5050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Prendergast G. C., Ziff E. B. Methylation-sensitive sequence-specific DNA binding by the c-Myc basic region. Science. 1991 Jan 11;251(4990):186–189. doi: 10.1126/science.1987636. [DOI] [PubMed] [Google Scholar]
  33. Propst F., Rosenberg M. P., Iyer A., Kaul K., Vande Woude G. F. c-mos proto-oncogene RNA transcripts in mouse tissues: structural features, developmental regulation, and localization in specific cell types. Mol Cell Biol. 1987 May;7(5):1629–1637. doi: 10.1128/mcb.7.5.1629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rappolee D. A., Brenner C. A., Schultz R., Mark D., Werb Z. Developmental expression of PDGF, TGF-alpha, and TGF-beta genes in preimplantation mouse embryos. Science. 1988 Sep 30;241(4874):1823–1825. doi: 10.1126/science.3175624. [DOI] [PubMed] [Google Scholar]
  35. Rebagliati M. R., Melton D. A. Antisense RNA injections in fertilized frog eggs reveal an RNA duplex unwinding activity. Cell. 1987 Feb 27;48(4):599–605. doi: 10.1016/0092-8674(87)90238-8. [DOI] [PubMed] [Google Scholar]
  36. Reuse S., Roger P. P., Vassart G., Dumont J. E. Enhancement of cmyc mRNA concentration in dog thyrocytes initiating DNA synthesis in response to thyrotropin, forskolin, epidermal growth factor and phorbol myristate ester. Biochem Biophys Res Commun. 1986 Dec 30;141(3):1066–1076. doi: 10.1016/s0006-291x(86)80152-8. [DOI] [PubMed] [Google Scholar]
  37. Sagata N., Oskarsson M., Copeland T., Brumbaugh J., Vande Woude G. F. Function of c-mos proto-oncogene product in meiotic maturation in Xenopus oocytes. Nature. 1988 Oct 6;335(6190):519–525. doi: 10.1038/335519a0. [DOI] [PubMed] [Google Scholar]
  38. Schmidt M., Oskarsson M. K., Dunn J. K., Blair D. G., Hughes S., Propst F., Vande Woude G. F. Chicken homolog of the mos proto-oncogene. Mol Cell Biol. 1988 Feb;8(2):923–929. doi: 10.1128/mcb.8.2.923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Spencer C. A., Groudine M. Control of c-myc regulation in normal and neoplastic cells. Adv Cancer Res. 1991;56:1–48. doi: 10.1016/s0065-230x(08)60476-5. [DOI] [PubMed] [Google Scholar]
  40. Stanton L. W., Watt R., Marcu K. B. Translocation, breakage and truncated transcripts of c-myc oncogene in murine plasmacytomas. Nature. 1983 Jun 2;303(5916):401–406. doi: 10.1038/303401a0. [DOI] [PubMed] [Google Scholar]
  41. Strickland S., Huarte J., Belin D., Vassalli A., Rickles R. J., Vassalli J. D. Antisense RNA directed against the 3' noncoding region prevents dormant mRNA activation in mouse oocytes. Science. 1988 Aug 5;241(4866):680–684. doi: 10.1126/science.2456615. [DOI] [PubMed] [Google Scholar]
  42. Studzinski G. P., Brelvi Z. S., Feldman S. C., Watt R. A. Participation of c-myc protein in DNA synthesis of human cells. Science. 1986 Oct 24;234(4775):467–470. doi: 10.1126/science.3532322. [DOI] [PubMed] [Google Scholar]
  43. Toulmé J. J., Hélène C. Antimessenger oligodeoxyribonucleotides: an alternative to antisense RNA for artificial regulation of gene expression--a review. Gene. 1988 Dec 10;72(1-2):51–58. doi: 10.1016/0378-1119(88)90127-8. [DOI] [PubMed] [Google Scholar]
  44. Varmus H. E. The molecular genetics of cellular oncogenes. Annu Rev Genet. 1984;18:553–612. doi: 10.1146/annurev.ge.18.120184.003005. [DOI] [PubMed] [Google Scholar]
  45. Weintraub H. M. Antisense RNA and DNA. Sci Am. 1990 Jan;262(1):40–46. doi: 10.1038/scientificamerican0190-40. [DOI] [PubMed] [Google Scholar]
  46. Weller A., Meek J., Adamson E. D. Preparation and properties of monoclonal and polyclonal antibodies to mouse epidermal growth factor (EGF) receptors: evidence for cryptic EGF receptors in embryonal carcinoma cells. Development. 1987 Jun;100(2):351–363. doi: 10.1242/dev.100.2.351. [DOI] [PubMed] [Google Scholar]
  47. Yokoyama K., Imamoto F. Transcriptional control of the endogenous MYC protooncogene by antisense RNA. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7363–7367. doi: 10.1073/pnas.84.21.7363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zimmerman K. A., Yancopoulos G. D., Collum R. G., Smith R. K., Kohl N. E., Denis K. A., Nau M. M., Witte O. N., Toran-Allerand D., Gee C. E. Differential expression of myc family genes during murine development. 1986 Feb 27-Mar 5Nature. 319(6056):780–783. doi: 10.1038/319780a0. [DOI] [PubMed] [Google Scholar]
  49. van der Krol A. R., Mol J. N., Stuitje A. R. Modulation of eukaryotic gene expression by complementary RNA or DNA sequences. Biotechniques. 1988 Nov-Dec;6(10):958–976. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES