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.
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References
Allen ND, Norris ML, Surani MA (1990) Epigenetic control of transgene expression and imprinting by genotype-specific modifiers. Cell 61:853–861
Breakefield XO (1993) Gene delivery into the brain using virus vectors. Nat Genet 3:187–189
Fire A (1992) Histochemical techniques for locating Escherichia coli beta-galactosidase activity in transgenic organisms. Genet Anal Tech Appl 9:151–158
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
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
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
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
Hogan B, Constantini F, Lacy E (1986) Manipulating the mouse embryo. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
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
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
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor, Laboratory, Cold Spring Harbor, NY
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
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
Paldi A, Deltour L, Jami J (1993) Cis effect of lacZ sequences in transgenic mice. Transgenic Res 2:325–329
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
Prost E, Moore DD (1986) CAT vectors for analysis of eukaryotic promoters and enhancers. Gene 45:107–111
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
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
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
Seed B, Sheen JY (1988) A simple phase-extraction assay for chloramphenicol acyltransferase activity. Gene 67:271–277
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
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
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
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
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
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
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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
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DOI: https://doi.org/10.1007/BF00269579