Skip to main content

Expression of Exogenous Genes in Xenopus Oocytes, Eggs, and Embryos

  • Protocol
  • 1562 Accesses

Part of the book series: Methods in Molecular Biology ((MIMB,volume 62))

Abstract

Molecular biology of gene expression in early amphibian embryogenesis began in 1964, when undegraded, as opposed to alkaline-hydrolyzed, PCA (perchloric acid), or TCA (trichloroacetic acid)-degraded, RNAs were extracted by Brown and Littna (13), Shiokawa and Yamana (4,5), and Woodland and Gurdon (6) from amphibian embryos using phenol methods. From these early studies, especially from those of Don Brown, it was realized that amphibian embryos exhibit quite unusual RNA synthetic patterns; the pattern of active 4S RNA (mainly tRNA) synthesis with no rRNA synthesis in pregastrula stages and the pattern of gradually increasing rRNA synthesis in post-gastrular stages. It was then reported in 1982 by Newport and Kirschner (7,8) that in Xenopus embryogenesis, large changes called midblastula transition (MBT) take place at the 12th cleavage, which include the appearance of Gl phase in cell cycle, onset of gene expression from zygotic nuclei, and acquisition of cellular motility (7,8). We then found in 1987 that Xenopus embryogenesis consists of three different phases with respect to the RNA synthetic pattern. The phase of a low level (on a per-embryo but not necessarily on a per-cell basis) of mRNA synthesis (pre-MBT stage), the phase of extremely active tRNA synthesis (both on per-cell and per-embryo bases) (MBT stage), and the phase of a nearly constant level (per cell) of rRNA synthesis (post-MBT stage) (912).

This is a preview of subscription content, log in via an institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Brown, D. D. and Littna, E. (1964) RNA synthesis during the development of Xenopus laevis, the South African clawed toad. J. Mol. Biol. 8, 669–687.

    Article  PubMed  CAS  Google Scholar 

  2. Brown, D. D. and Littna, E. (1966) Synthesis and accumulation of DNA-like RNA during embryogenesis of Xenopus laevis. J. Mol. Biol. 20, 81–94.

    Article  PubMed  CAS  Google Scholar 

  3. Brown, D. D. and Littna, E. (1966) Synthesis and accumulation of low molecular weight RNA during embryogenesis of Xenopus laevis. J. Mol. Biol. 20, 95–112.

    Article  PubMed  CAS  Google Scholar 

  4. Shiokawa, K. and Yamana, K. (1965) Demonstration of “polyphosphate” and its possible role in RNA synthesis during early development of Rana japonica embryos. Exptl. Cell Res. 38, 180–186.

    Article  PubMed  CAS  Google Scholar 

  5. Shiokawa, K. and Yamana, K. (1967) Pattern of RNA synthesis in isolated cells of Xenopus laevis embryos. Dev. Biol. 368–388.

    Google Scholar 

  6. Woodland, H. R. and Gurdon, J. B. (1968) The relative rates of synthesis of DNA sRNA and rRNA in the endodermal region and other parts of Xenopus laevis embryos. J. Embryol. Exp. Morph. 19, 363–385.

    PubMed  CAS  Google Scholar 

  7. Newport, J. and Kirschner, M. (1982) A major developmental transition in early Xenopus embryos: I. Characterization and timing of cellular changes at the midblastula stage. Cell 30, 675–686.

    Article  PubMed  CAS  Google Scholar 

  8. Newport, J. and Kirschner, M. (1982) A major developmental transition in early Xenopus embryos: II. Control of the onset of transcription. Cell 30, 687–696.

    Article  PubMed  CAS  Google Scholar 

  9. Shiokawa, K., Misumi, Y., and Yamana, K. (1981) Demonstration of rRNA synthesis in pre-gastrular embryos of Xenopus laevis. Dev. Growth Differ. 23, 579–587.

    Article  CAS  Google Scholar 

  10. Shiokawa, K., Tashiro, K., Misumi, Y., and Yamana, K. (1981) Non-coordinated synthesis of RNAs in pre-gastrular embryos of Xenopus laevis. Dev. Growth Differ. 23, 589–597.

    Article  CAS  Google Scholar 

  11. Shiokawa, K. (1991) Gene expression from endogenous and exogenously-introduced DNAs in early embryogenesis of xenopus laevis. Dev. Growth Differ. 33, 1–8.

    Article  CAS  Google Scholar 

  12. Shiokawa, K., Kurashima, R., and Shinga, J. (1994) Temporal control of gene expression from endogenous and exogenously-introduced DNAs in early embryo-genesis of Xenopus laevis. Int. J. Dev. Biol. 38, 249–255.

    PubMed  CAS  Google Scholar 

  13. Gurdon, J. B. and Melton, D. A. (1981) Gene transfer in amphibian eggs and oocytes. Ann. Rev. Genet. 15, 189–218.

    Article  PubMed  CAS  Google Scholar 

  14. Gurdon, J. B. and Brown, D. D. (1977) DNA microinjection, in The Molecular Biology of the Genetic Apparatus, vol. 2 (T’so, P., ed.), North-Holland, Amsterdam, pp. 111–123.

    Google Scholar 

  15. Asano, M. and Shiokawa, K. (1993) Behavior of exogenously-injected DNAs in early embryos of Xenopus laevis. Zool. Sci. 10, 197–222.

    CAS  Google Scholar 

  16. Dumont, J. N. (1972) Oogenesis in Xenopus laevis: I. Stages of oocyte development in laboratory maintained animals. J. Morphol. 136, 153–180.

    Article  PubMed  CAS  Google Scholar 

  17. Shiokawa, K., Nada, O., and Yamana, K. (1967) RNA synthesis in isolated cells from Xenopus laevis embryos. Nature 213, 1027, 1028.

    Article  CAS  Google Scholar 

  18. Shiokawa, K., Fu, Y., Nakakura, N., Tashiro, K., Sameshima, M., and Hosokawa, K. (1989) Effects of the injection of exogenous DNAs on gene expression in early embryos and coenocytic egg cells of Xenopus laevis Roux’s Arch. Dev. Biol. 198, 78–84.

    Article  CAS  Google Scholar 

  19. Lund, E. and Dahlberg, J. E. (1992) Control of 4-8S RNA transcription at the midblastula transition in Xenopus laevis embryos. Genes Dev. 6, 1097–1106.

    Article  PubMed  CAS  Google Scholar 

  20. Shiokawa, K., Yamana, K., Fu, Y., Atsuchi, Y., and Hosokawa, K. (1990) Expression of exogeneously introduced bacterial chloramphenicol acetyl-transferase genes in Xenopus laevis embryos before the midblastula transition. Roux’s Arch. Dev. Biol. 198, 322–329.

    Article  CAS  Google Scholar 

  21. Dawid, I. B. (1966) Deoxyribonucleic acid in amphibian eggs. J. Mol. Biol. 12, 581–599.

    Article  Google Scholar 

  22. Harvey, R. P. and Melton, D. A. (1988) Microinjection of synthetic Xhox-1A homeobox mRNA disrupts somite formation in developing Xenopus embryos. Cell 53, 687–697.

    Article  PubMed  CAS  Google Scholar 

  23. Amaya, E., Musci, T. J., and Kirschner, M. W. (1991) Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos. Cell 66, 257–270.

    Article  PubMed  CAS  Google Scholar 

  24. Nieuwkoop, P. D. and Faber, J. (1956) Normal Table of Xenopus laevis (Daudin) North Holland, Amsterdam.

    Google Scholar 

  25. Shiokawa, K., Tashiro, K., Yamana, K., and Sameshima, M. (1987) Electron microscopic studies of giant nucleus-like structure formed by lambda DNA introduced into the cytoplasm of Xenopus laevis fertilized eggs and embryos. Cell Differ. 20, 253–261.

    Article  PubMed  CAS  Google Scholar 

  26. Shiokawa, K., Yoshida, M., Fukamachi, H., Fu, Y., Tashiro, K., and Sameshima, M. (1992) Cytological studies of large nucleus-like structures formed by exogenously-injected linear and circular DNAs in fertilized eggs of Xenopus laevis. Dev. Growth Differ. 34, 79–90.

    Article  Google Scholar 

  27. Forbes, D. J., Kirschner, M. W., and Newport, J. W. (1983) Spontaneous formation of nucleus-like structures around bacteriophage DNA microinjected into Xenopus eggs. Cell 34, 13–23.

    Article  PubMed  CAS  Google Scholar 

  28. Shiokawa, K., Sameshima, M., Tashiro, K., Miura, T., Nakakura, N., and Yamana, K. (1986) Formation of nucleus-like structure in the cytoplasm of lambda-DNA-injected fertilized eggs and its partition into blastomeres during early embryogenesis in Xenopus laevis. Dev. Biol. 116, 539–542.

    Article  PubMed  CAS  Google Scholar 

  29. Trendelenburg, M. F., Oudet, P., Spring, H., and Montag, M. (1986) DNA injections into Xenopus embryos fate of injected DNA in relation to formation of embryonic nuclei. J. Embryol. Exp. Morph. 97 (Suppl.), 243–255.

    Google Scholar 

  30. Hofmann, A., Montag, M., Steinbeisser, H., and Trendelenburg, F. (1990) Plasmid and bacteriophage lambda-DNA show differential replication characteristics following injection into fertilized eggs of Xenopus laevis dependence on period and site of injection. Cell Differ. Dev. 30, 77–85.

    Article  PubMed  CAS  Google Scholar 

  31. Etkin, L. and Pearman, B. (1987) Distribution, expression and germ line transmission of exogenous DNA sequences following microninjection into Xenopus laevis eggs. Development 99, 15–23.

    PubMed  CAS  Google Scholar 

  32. Andres A.-C., Muellner, D. B., and Ryffel, G. U. (1984) Persistence, methylation and expression of vitellogenin gene derivatives after injection into fertilized eggs of Xenopus laevis. Nucleic Acids Res. 12, 2283–2302.

    Article  PubMed  CAS  Google Scholar 

  33. Fu, Y., Hosokawa, K., and Shiokawa, K. (1989) Expression of circular and linearized bacterial chloramphenicol acetyltransferase genes with or without viral promoters after in injection into fertilized eggs, unfertilized eggs and oocytes of Xenopus laevis. Rouxs Arch. Dev. Biol. 198, 148–156.

    Article  CAS  Google Scholar 

  34. Shiokawa, K., Yamazaki, T., Fu, Y., Tashiro, K., Tsurugi, K., Motizuki, M., Ikegami, Y., Araki, E., Andoh, T., and Hosokawa, K. (1989b) Persistence and expression of circular DNAs encoding Drosophila amylase, bacterial chloramphenicol acetyltransferase, and others in Xenopus laevis embryos. Cell Struct. Func. 14, 261–269.

    Article  CAS  Google Scholar 

  35. Harland, R. M. and Laskey, R. A. (1980) Regulated replication of DNA microinjected into eggs of Xenopus laevis. Cell 21, 761–771.

    Article  PubMed  CAS  Google Scholar 

  36. Mechali, M. and Kearsey, S. (1984) Lack of specific sequence requirement for DNA replication in Xenopus eggs compared with high sequence specificity in yeast. Cell 38, 55–64.

    Article  PubMed  CAS  Google Scholar 

  37. Shiokawa, K., Fu, Y., Hosokawa, K., and Yamana, K. (1990) Temporally uncontrolled expression of linearized plasmid DNA which carries bacterial chloramphenicol acetyltransferase gene with Xenopus cardiac a-actin promoter after injection into Xenopus fertilized eggs. Roux’s Arch. Dev. Biol. 199, 171–180.

    Article  Google Scholar 

  38. Bendig, M. M. (1981) Persistence and expression of histone genes injected into Xenopus eggs in early development. Nature 292, 65–67.

    Article  PubMed  CAS  Google Scholar 

  39. Bendig, M. M. and Williams, J. G. (1984) Differential expression of the Xenopus laevis tadpole and adult b-globin genes when injected into fertilized Xenopus laevis eggs. Mol. Cell Biol. 4, 567–570.

    PubMed  CAS  Google Scholar 

  40. Rusconi, S. and Schaffner, W. (1981) Transformation of frog embryos with a rabbit beta-globin gene. Proc. Natl. Acad. Sci. USA 78, 5051–5055.

    Article  PubMed  CAS  Google Scholar 

  41. Etkin, L. D., Pearman, B., Roberts, M., and Bektesh, S. (1984) Replication, integration and expression of exogenous DNA injected into fertilized eggs of Xenopus laevis. Differentiation 26, 191–202.

    Article  Google Scholar 

  42. Bendig, M. M. and Williams, J. G. (1984) Fidelity of transcription of Xenopus laevis globin genes injected into Xenopus laevis oocytes and unfertilized eggs. Mol. Cell Biol. 4, 2109–2119.

    PubMed  CAS  Google Scholar 

  43. Fu, Y., Sato, K., Hosokawa, K., and Shiokawa, K. (1990) Expression of circular plasmids which contain bacterial chloramphenicol acetyltransferase gene connected to the promoter of polypeptide IX gene of human adenovirus type 12 in oocytes, eggs and embryos of Xenopus laevis. Zool. Sci. 7, 195–200.

    CAS  Google Scholar 

  44. Harland, R. M., Weintraub, H., and McKnight, S. L. (1983) Transcription of DNA injected into Xenopus oocytes is influenced by template topology. Nature 302, 38–43.

    Article  PubMed  CAS  Google Scholar 

  45. Probst, E., Kressmann, A., and Birnstiel, M. L. (1979) Expression of sea urchin histone genes in the oocyte of Xenopus laevis. J. Mol. Biol. 135, 709–732.

    Article  PubMed  CAS  Google Scholar 

  46. Pruitt, S. and Reeder, R. H. (1984) Effect of topological constraint on transcription of ribosomal DNA in Xenopus oocytes. Comparison of plasmid and endogenous genes. J. Mol. Biol. 174, 121–139.

    Article  PubMed  CAS  Google Scholar 

  47. Roeder, R. G. (1974) Multiple forms of deoxyribonucleic acid-dependent ribonucleic acid polymerase in Xenopus laevis. Levels of activity during oocyte and embryonic development. J. Biol. Chem. 249, 249–256.

    PubMed  CAS  Google Scholar 

  48. Wickens, in P., Woo, S., O’Malley, B. W., and Gurdon, J. B. (1980) Expression of a chicken chromosomal ovalbumin gene injected into frog oocyte nuclei. Nature 285, 628–634.

    Article  PubMed  CAS  Google Scholar 

  49. Wilson, C., Cross, G. S., and Woodland, H. R. (1986) Tissue-specific expression of actin genes injected into Xenopus laevis. Cell 47, 589–599.

    Article  PubMed  CAS  Google Scholar 

  50. Steinbeisser, H., Alonso, A., Epperlein, H.-H., and Trendelenburg, M. F. (1989) Expression of mouse histone H1(o) promoter sequences following microinjection into Xenopus oocytes and developing embryos. Int. J. Dev. Biol. 33, 361–368.

    PubMed  CAS  Google Scholar 

  51. Mohun, T. J., Garrett, N., and Gurdon, J. B. (1986) Upstream sequences required for tissue-specific activation of the cardiac actin gene in Xenopus laevis embryos. EMBO J. 5, 3185–3193.

    PubMed  CAS  Google Scholar 

  52. Steinbeisser, H., Hofmann, A., Stutz, F., and Trendelenburg, M. F. (1988) Different regulatory elements are required for cell-type and stage-specific expression of the Xenopus laevis skeletal muscle actin gene upon injection X. laevis oocytes and embryos. Nucleic Acids Res. 16, 3223–3238.

    Article  PubMed  CAS  Google Scholar 

  53. Brennan, S. M. (1990) Transcription of endogenous and injected cytoskeletal actin genes during early embryonic development in Xenopus laevis. Differentiation 44, 111–121.

    Article  PubMed  CAS  Google Scholar 

  54. Jonas, E. A., Snape, A. M., and Sargent, T. D. (1989) Transcriptional regulation of a Xenopus embryonic epidermal keratin gene. Development 106, 399–405.

    PubMed  CAS  Google Scholar 

  55. Krieg, P. A. and Melton, D. A. (1987) An enhancer responsible for activating transcription at the mid-blastula transition in Xenopus development. Proc. Natl. Acad. Sci. USA 84, 2331–2335.

    Article  PubMed  CAS  Google Scholar 

  56. Krone, P. H. and Heikkila, J. J. (1989) Expression of microinjected hsp70/CAT and hsp30/CAT chimeric genes in developing Xenopus laevis embryos. Development 106, 271–281.

    PubMed  CAS  Google Scholar 

  57. Winning, R. S., Bols, N. C., Wooden, S. K., Lee, A. S., and Heikkila, J. J. (1992) Analysis of the expression of a glucose-regulated protein (GRP78) promoter/CAT fusion gene during early Xenopus laevis development. Differentiation 49, 1–6.

    Article  PubMed  CAS  Google Scholar 

  58. Shiokawa, K., Tashiro, K., Kurashima, R., and Amano, M. (1994) Frogs as the laboratory animal for exogenous gene introduction, in Mouse as a Laboratory Animal. (Yamamura, K., Katsuki, M., and Aizawa, S., eds.), Nakayamashoten, Japan (In Japanese), p. 279.

    Google Scholar 

  59. Nakakura, N., Miura, T., Yamana, K., Ito, A., and Shiokawa, K. (1987) Synthesis of heterogeneous mRNA-like RNA and low-molecular-weight RNA before the midblastula transition in embryos of Xenopus laevis. Dev. Biol. 123, 421–429.

    Article  PubMed  CAS  Google Scholar 

  60. Shiokawa, K., Misumi, Y., Tashiro, K., Nakakura, N., Yamana, K., and Oh-uchida, M. (1989) Changes in the patterns of RNA synthesis in early embryogenesis of Xenopus laevis. Cell Differ. Dev. 28, 17–26.

    Article  PubMed  CAS  Google Scholar 

  61. Etkin, L. D. and Balcells, S. (1985) Transformed Xenopus embryos as a transient expression system to analyze gene expression at the midblastula transition. Dev. Biol. 108, 173–178.

    Article  PubMed  CAS  Google Scholar 

  62. Johnson, A. D. and Krieg, P. A. (1994) pXeX, a vector for efficient expression of cloned sequences in Xenopus embryos. Gene 147, 223–226.

    Article  PubMed  CAS  Google Scholar 

  63. Vezina, C., Wooden, S. K., Lee, A. S., and Heikkile, J. J. (1994) Constitutive expression of a microinjected glucose-regulated protein (grp78) fusion gene during early Xenopus laevis development. Differentiation 57, 171–177.

    Article  PubMed  CAS  Google Scholar 

  64. Busby, S. J. and Reeder, R. H. (1983) Spacer sequences regulate transcription of ribosomal gene plasmids injected into Xenopus embryos. Cell 34, 989–996.

    Article  PubMed  CAS  Google Scholar 

  65. Honjo, T. and Reeder, R. H. (1973) Preferential transcription of Xenopus laevis ribosomal RNA in interspecies hybrids between Xenopus laevis and Xenopus mulleri. J. Mol. Biol. 80, 217–228.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 1997 Humana Press Inc.

About this protocol

Cite this protocol

Shiokawa, K., Koga, C., Ito, Y., Shibata, M. (1997). Expression of Exogenous Genes in Xenopus Oocytes, Eggs, and Embryos. In: Tuan, R.S. (eds) Recombinant Gene Expression Protocols. Methods in Molecular Biology, vol 62. Humana Press. https://doi.org/10.1385/0-89603-480-1:247

Download citation

  • DOI: https://doi.org/10.1385/0-89603-480-1:247

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-480-8

  • Online ISBN: 978-1-59259-548-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics