Skip to main content
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
. 1995 Feb 28;92(5):1317–1321. doi: 10.1073/pnas.92.5.1317

Real time imaging of transcriptional activity in live mouse preimplantation embryos using a secreted luciferase.

E M Thompson 1, P Adenot 1, F I Tsuji 1, J P Renard 1
PMCID: PMC42510  PMID: 7877974

Abstract

Study of gene expression kinetics during preimplantation mammalian development is difficult because of the limited amount of material and the usually destructive, static nature of molecular analyses. We describe continuous, noninvasive monitoring of gene expression in preimplantation embryos by using a secreted luminescent reporter, Vargula luciferase. Transgene expression profiles were followed by assaying aliquots of culture medium or by direct visualization of Vargula luciferase secretion from living embryos in real time through photon imaging. With this approach, it is possible to observe epigenetic modulations of gene expression and to link this over time to the developmental capacity of individual embryos. In addition, by developing a strategy where expression from integrated transgenes is enhanced relative to that from nonintegrated DNA, we provide evidence that rapid detection of transgene integration prior to the blastocyst stage should be possible. Thus, imaging of Vargula luciferase secretion may also be useful in the early screening of embryos, for example, in the production of transgenic livestock.

Full text

PDF
1317

Images in this article

Selected References

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

  1. Anzai M., Nakagata N., Matsumoto K., Takahashi A., Takahash Y., Miyata K. [Cryopreservation of in vitro fertilized embryos from transgenic rat by ultrarapid freezing]. Jikken Dobutsu. 1994 Apr;43(2):247–250. [PubMed] [Google Scholar]
  2. Bensaude O., Babinet C., Morange M., Jacob F. Heat shock proteins, first major products of zygotic gene activity in mouse embryo. Nature. 1983 Sep 22;305(5932):331–333. doi: 10.1038/305331a0. [DOI] [PubMed] [Google Scholar]
  3. Bode J., Maass K. Chromatin domain surrounding the human interferon-beta gene as defined by scaffold-attached regions. Biochemistry. 1988 Jun 28;27(13):4706–4711. doi: 10.1021/bi00413a019. [DOI] [PubMed] [Google Scholar]
  4. Bowen R. A., Reed M. L., Schnieke A., Seidel G. E., Jr, Stacey A., Thomas W. K., Kajikawa O. Transgenic cattle resulting from biopsied embryos: expression of c-ski in a transgenic calf. Biol Reprod. 1994 Mar;50(3):664–668. doi: 10.1095/biolreprod50.3.664. [DOI] [PubMed] [Google Scholar]
  5. Craig F. F., Simmonds A. C., Watmore D., McCapra F., White M. R. Membrane-permeable luciferin esters for assay of firefly luciferase in live intact cells. Biochem J. 1991 Jun 15;276(Pt 3):637–641. doi: 10.1042/bj2760637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. DiLella A. G., Hope D. A., Chen H., Trumbauer M., Schwartz R. J., Smith R. G. Utility of firefly luciferase as a reporter gene for promoter activity in transgenic mice. Nucleic Acids Res. 1988 May 11;16(9):4159–4159. doi: 10.1093/nar/16.9.4159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gasser S. M., Amati B. B., Cardenas M. E., Hofmann J. F. Studies on scaffold attachment sites and their relation to genome function. Int Rev Cytol. 1989;119:57–96. doi: 10.1016/s0074-7696(08)60649-x. [DOI] [PubMed] [Google Scholar]
  8. Getzenberg R. H., Pienta K. J., Ward W. S., Coffey D. S. Nuclear structure and the three-dimensional organization of DNA. J Cell Biochem. 1991 Dec;47(4):289–299. doi: 10.1002/jcb.240470402. [DOI] [PubMed] [Google Scholar]
  9. Goring D. R., Rossant J., Clapoff S., Breitman M. L., Tsui L. C. In situ detection of beta-galactosidase in lenses of transgenic mice with a gamma-crystallin/lacZ gene. Science. 1987 Jan 23;235(4787):456–458. doi: 10.1126/science.3099390. [DOI] [PubMed] [Google Scholar]
  10. Gurdon J. B. The generation of diversity and pattern in animal development. Cell. 1992 Jan 24;68(2):185–199. doi: 10.1016/0092-8674(92)90465-o. [DOI] [PubMed] [Google Scholar]
  11. Horvat S., Medrano J. F., Behboodi E., Anderson G. B., Murray J. D. Sexing and detection of gene construct in microinjected bovine blastocysts using the polymerase chain reaction. Transgenic Res. 1993 May;2(3):134–140. doi: 10.1007/BF01972606. [DOI] [PubMed] [Google Scholar]
  12. Hou Q., Gorski J. Estrogen receptor and progesterone receptor genes are expressed differentially in mouse embryos during preimplantation development. Proc Natl Acad Sci U S A. 1993 Oct 15;90(20):9460–9464. doi: 10.1073/pnas.90.20.9460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hunt C., Calderwood S. Characterization and sequence of a mouse hsp70 gene and its expression in mouse cell lines. Gene. 1990 Mar 15;87(2):199–204. doi: 10.1016/0378-1119(90)90302-8. [DOI] [PubMed] [Google Scholar]
  14. Inouye S., Ohmiya Y., Toya Y., Tsuji F. I. Imaging of luciferase secretion from transformed Chinese hamster ovary cells. Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9584–9587. doi: 10.1073/pnas.89.20.9584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Klehr D., Maass K., Bode J. Scaffold-attached regions from the human interferon beta domain can be used to enhance the stable expression of genes under the control of various promoters. Biochemistry. 1991 Feb 5;30(5):1264–1270. doi: 10.1021/bi00219a015. [DOI] [PubMed] [Google Scholar]
  16. Overbeek P. A., Aguilar-Cordova E., Hanten G., Schaffner D. L., Patel P., Lebovitz R. M., Lieberman M. W. Coinjection strategy for visual identification of transgenic mice. Transgenic Res. 1991 Dec;1(1):31–37. doi: 10.1007/BF02512994. [DOI] [PubMed] [Google Scholar]
  17. Phi-Van L., von Kries J. P., Ostertag W., Strätling W. H. The chicken lysozyme 5' matrix attachment region increases transcription from a heterologous promoter in heterologous cells and dampens position effects on the expression of transfected genes. Mol Cell Biol. 1990 May;10(5):2302–2307. doi: 10.1128/mcb.10.5.2302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pursel V. G., Rexroad C. E., Jr Status of research with transgenic farm animals. J Anim Sci. 1993;71 (Suppl 3):10–19. doi: 10.2527/1993.71suppl_310x. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Shimomura O., Johnson F. H., Masugi T. Cypridina bioluminescence: light-emitting oxyluciferin-luciferase complex. Science. 1969 Jun 13;164(3885):1299–1300. doi: 10.1126/science.164.3885.1299. [DOI] [PubMed] [Google Scholar]
  21. Thompson E. M., Nagata S., Tsuji F. I. Cloning and expression of cDNA for the luciferase from the marine ostracod Vargula hilgendorfii. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6567–6571. doi: 10.1073/pnas.86.17.6567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Thompson E. M., Nagata S., Tsuji F. I. Vargula hilgendorfii luciferase: a secreted reporter enzyme for monitoring gene expression in mammalian cells. Gene. 1990 Dec 15;96(2):257–262. [PubMed] [Google Scholar]
  23. Yang J., Thomason D. B. An easily synthesized, photolyzable luciferase substrate for in vivo luciferase activity measurement. Biotechniques. 1993 Nov;15(5):848–850. [PubMed] [Google Scholar]
  24. Yoshida M., Kijima M., Akita M., Beppu T. Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A. J Biol Chem. 1990 Oct 5;265(28):17174–17179. [PubMed] [Google Scholar]
  25. Zhao K., Käs E., Gonzalez E., Laemmli U. K. SAR-dependent mobilization of histone H1 by HMG-I/Y in vitro: HMG-I/Y is enriched in H1-depleted chromatin. EMBO J. 1993 Aug;12(8):3237–3247. doi: 10.1002/j.1460-2075.1993.tb05993.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. de Wet J. R., Wood K. V., DeLuca M., Helinski D. R., Subramani S. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987 Feb;7(2):725–737. doi: 10.1128/mcb.7.2.725. [DOI] [PMC free article] [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