Skip to main content

Improved in situ β-galactosidase staining for histological analysis of transgenic mice

Abstract

The present study describes a novel method for the histochemical demonstration of β-galactosidase activity on tissue sections. We have replaced 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-Gal) with 5-bromoindolyl-β-o-galactopyranoside (Bluo-Gal) as a chromogenic substrate for the bacterial β-galactosidase (lacZ). After β-galactosidic cleavage, Bluo-Gal precipitates in form of fine birefringent crystals, whereas X-gal gives rise to an amorphous precipitate. Upon microscopic examination under polarized light, the crystals emit a strong signal consisting of yellow reflected light. This property of Bluo-Gal results in greatly enhanced sensitivity of the staining method for β-galactosidase and allows for optimal morphological resolution. To exemplify the applications of this technique, the expression is demonstrated in transgenic mice of β-galactosidase driven by a fragment of the human tissue-type plasminogen activator promoter.

This is a preview of subscription content, access via your institution.

References

  1. Allen ND, Norris ML, Surani MA (1990) Epigenetic control of transgene expression and imprinting by genotype-specific modifiers. Cell 61:853–861

    Google Scholar 

  2. Breakefield XO (1993) Gene delivery into the brain using virus vectors. Nat Genet 3:187–189

    Google Scholar 

  3. Fire A (1992) Histochemical techniques for locating Escherichia coli beta-galactosidase activity in transgenic organisms. Genet Anal Tech Appl 9:151–158

    Google Scholar 

  4. Frank E, Sanes JR (1991) Lineage of neurons and glia in chick dorsal root ganglia: analysis in vivo with a recombinant retrovirus. Development 111:895–908

    Google Scholar 

  5. Friedrich G, Soriano P (1991) Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice. Genes Dev 5:1513–1523

    Google Scholar 

  6. Guzman RJ, Lemarchand P, Crystal RG, Epstein SE, Finkel T (1993) Efficient and selective adenovirus-mediated gene transfer into vascular neointima. Circulation 88:2838–2848

    Google Scholar 

  7. Hall CV, Jacob PE, Ringold GM, Lee F (1983) Expression and regulation of Escherichia coli lacZ gene fusions in mammalian cells. J Mol Appl Genet 2:101–109

    Google Scholar 

  8. Hogan B, Constantini F, Lacy E (1986) Manipulating the mouse embryo. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

    Google Scholar 

  9. Le Mouellic H, Lallemand Y, Brulet P (1990) Targeted replacement of the homebox gene Hox-3.1 by the Escherichia coli lacZ in mouse chimeric embryos. Proc Natl Acad Sci USA 87:4712–4716

    Google Scholar 

  10. MacGregor GR, Mogg AE, Burke JF, Caskey CT (1987) Histochemical staining of clonal mammalian cell lines expressing E. coli beta galactosidase indicates heterogeneous expression of the bacterial gene. Somat Cell Mol Genet 13:253–265

    Google Scholar 

  11. Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor, Laboratory, Cold Spring Harbor, NY

    Google Scholar 

  12. Miller JH (1978) The lacI gene: its role in lac operon control and its use as a genetic system. In: Miller JH (ed) The operon. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp 31–88

    Google Scholar 

  13. Mucke L, Oldstone MB, Morris JC, Nerenberg MI (1991) Rapid activation of astrocyte-specific expression of GFAP-lacZ transgene by focal injury. New Biol 3:465–474

    Google Scholar 

  14. Paldi A, Deltour L, Jami J (1993) Cis effect of lacZ sequences in transgenic mice. Transgenic Res 2:325–329

    Google Scholar 

  15. Price J, Turner D, Cepko C (1987) Lineage analysis in the vertebrate nervous system by retrovirus-mediated gene transfer. Proc Natl Acad Sci USA 84:156–160

    Google Scholar 

  16. Prost E, Moore DD (1986) CAT vectors for analysis of eukaryotic promoters and enhancers. Gene 45:107–111

    Google Scholar 

  17. Renucci A, Zappavigna V, Zakany J, Izpisua Belmonte JC, Burki K, Duboule D (1992) Comparison of mouse and human HOX-4 complexes defines conserved sequences involved in the regulation of Hox-4.4. EMBO J 11:1459–1468

    Google Scholar 

  18. Rosenberg WS, Breakefield XO, DeAntonio C, Isacson O (1992) Authentic and artifactual detection of the E. coli lacZ gene product in the rat brain by histochemical methods. Brain Res Mol Brain Res 16:311–315

    Google Scholar 

  19. Schneider Maunoury S, Topilko P, Seitandou T, Levi G, Cohen Tannoudji M, Pournin S, Babinet C, Charnay P (1993) Disruption of Krox-20 results in alteration of rhombomeres 3 and 5 in the developing hindbrain. Cell 75:1199–1214

    Google Scholar 

  20. Seed B, Sheen JY (1988) A simple phase-extraction assay for chloramphenicol acyltransferase activity. Gene 67:271–277

    Google Scholar 

  21. Sham MH, Hunt P, Nonchev S, Papalopulu N, Graham A, Boncinelli E, Krumlauf R (1992) Analysis of the murine Hox-2.7 gene: conserved alternative transcripts with differential distributions in the nervous system and the potential for shared regulatory regions. EMBO J 11:1825–1836

    Google Scholar 

  22. Smeyne RJ, Schilling K, Robertson L, Luk D, Oberdick J, Curran T, Morgan JI (1992) Fos-lacZ transgenic mice: mapping sites of gene induction in the central nervous system. Neuron 8:13–23

    Google Scholar 

  23. Smeyne RJ, Schilling K, Oberdick J, Robertson L, Luk D, Curran T, Morgan JI (1993a) A fos-lacZ transgenic mouse that can be used for neuroanatomic mapping. Adv Neurol 59:285–291

    Google Scholar 

  24. Smeyne RJ, Vendrell M, Hayward M, Baker SJ, Miao GG, Schilling K, Robertson LM, Curran T, Morgan JI (1993b) Continuous c-fos expression precedes programmed cell death in vivo. Nature 363:166–169

    Google Scholar 

  25. Wiestler OD, Aguzzi A, Schneemann M, Eibl R, Von Deimling A, Kleihues P (1992a) Oncogene complementation in fetal brain transplants. Cancer Res 52:3760–3767

    Google Scholar 

  26. Wiestler OD, Brustle O, Eibl RH, Radner H, Aguzzi A, Kleihues P (1992b) Retrovirus-mediated oncogene transfer into neural transplants. Brain Pathol 2:47–59

    Google Scholar 

Download references

Author information

Affiliations

Authors

Corresponding author

Correspondence to A. Aguzzi.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Aguzzi, A., Theuring, F. Improved in situ β-galactosidase staining for histological analysis of transgenic mice. Histochemistry 102, 477–481 (1994). https://doi.org/10.1007/BF00269579

Download citation

Keywords

  • Transgenic Mouse
  • Tissue Section
  • Plasminogen
  • Activator Promoter
  • Plasminogen Activator