Whole Mount Enzyme Histochemistry as a Rapid Screen at Necropsy for Expression of β-Galactosidase ( LacZ )–Bearing Transgenes: Considerations for Separating Specific LacZ Activity from Nonspecific (Endogenous) Galactosidase Activity
Abstract
Whole mount enzyme histochemistry to localize lacZ-bearing transgenes (lacZ-WMH) also detects endogenous β-galactosidases. The experiments reported here evaluated lacZ-WMH as a potential tool for transgene expression analysis during high-throughput rodent necropsies. A lacZ-WMH survey of organs from adult, wild-type, male and female mice (C57BL/6, FVB/N) and female rats (Sprague-Dawley) performed at the optimal pH (≥ 7.0) for bacterial lacZ yielded intense endogenous staining in the gonads, kidney, male accessory sex organs, salivary glands, submucosal glands in the duodenum, and thyroid. Substantial staining occurred in the adrenal cortex, lymph nodes, and linings of the gastrointestinal tract, the urinary bladder and uterus, and (for rat only) in the adenohypophysis, bone marrow, thymus, and trigeminal ganglia. Endogenous galactosidases were distributed similarly in sections of flash-frozen organs used for slide-based lacZ histochemistry (lacZ-SBH) at pH ≤ 5.0 (optimal for eukaryotic enzymes). Cerebral neurons were labeled only by lacZ-SBH. At pH 7.4, endogenous but not specific lacZ activity was abolished for lacZ-SBH, while endogenous activity was not halted without reducing specific activity for lacZ-WMH. These data demonstrate that lacZ-WMH is feasible during rodent necropsies for many but not all organs if species-, strain-, and sex-specific divergence in endogenous galactosidase activity is considered and special fixation (3% paraformaldehyde for 3 hours at 4°C) is used.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
Elevated endogenous β-gal activity can be found in some non-senescent cells.
“elevated endogenous β-gal activity can also be found in some non-senescent cells.48,49”
From Elimination of senescent cells by β-galactosidase-targeted prodrug attenuates inflammation and restores physical function in aged mice (2020) · cites this paper as [49]
Cites
The rat brain in stereotaxic coordinates
Palkovits, M. (1983) · Neuropeptides · doi:10.1016/0143-4179(83)90049-5 · Cited by 28,959 · see citing works
The Rat Brain in Stereotaxic Coordinates
Paxinos, G., & Watson, C. (1983) · Elsevier eBooks · doi:10.1016/c2009-0-63235-9 · Cited by 28,585 · see citing works
Friedrich, G., & Soriano, P. M. (1991) · Genes & Development · doi:10.1101/gad.5.9.1513 · Cited by 1,403 · see citing works
Subregion- and Cell Type–Restricted Gene Knockout in Mouse Brain
Tsien, J. Z., Chen, D. F., Gerber, D., Tom, C., Mercer, E. H., Anderson, D. J., Mayford, M., Kandel, E. R., & Tonegawa, S. (1996) · Cell · doi:10.1016/s0092-8674(00)81826-7 · Cited by 1,280 · see citing works
GERM-LINE TRANSFORMATION OF MICE
Palmiter, R. D., & Brinster, R. L. (1986) · Annual Review of Genetics · doi:10.1146/annurev.ge.20.120186.002341 · Cited by 1,157 · see citing works
Use of a recombinant retrovirus to study post‐implantation cell lineage in mouse embryos.
Sanes, J. R., Rubenstein, J. L. R., & Nicolas, J. F. (1986) · The EMBO Journal · doi:10.1002/j.1460-2075.1986.tb04620.x · Cited by 1,116 · see citing works
Reporter genes: Application to the study of mammalian gene transcription
Alam, J., & Cook, J. L. (1990) · Analytical Biochemistry · doi:10.1016/0003-2697(90)90601-5 · Cited by 471 · see citing works
Jain, V. K., & Magrath, I. T. (1991) · Analytical Biochemistry · doi:10.1016/0003-2697(91)90278-2 · Cited by 219 · see citing works
Expression of Bacterial β-Galactosidase in Animal Cells
An, G., Hidaka, K., & Siminovitch, L. (1982) · Molecular and Cellular Biology · doi:10.1128/mcb.2.12.1628 · Cited by 176 · see citing works
HISTOCHEMICAL DEMONSTRATION OF β-D-GALACTOSIDASE IN THE RAT
Rutenburg, A. M., Rutenburg, S. H., Monis, B., et al. (1958) · Journal of Histochemistry & Cytochemistry · doi:10.1177/6.2.122 · Cited by 166 · see citing works
Spergel, D. J., Krüth, U., Shimshek, D. R., Sprengel, R., & SEEBURG, P. H. (2001) · Progress in Neurobiology · doi:10.1016/s0301-0082(00)00038-1 · Cited by 163 · see citing works
Use of E. coli lacZ (β-Galactosidase) as a Reporter Gene
MacGregor, G. R., Nolan, G. P., Fiering, S., Roederer, M., & Herzenberg, L. A. (2003) · Humana Press eBooks · doi:10.1385/0-89603-178-0:217 · Cited by 147 · see citing works
Gow, A., Friedrich, V. L., & Lazzarini, R. A. (1992) · The Journal of Cell Biology · doi:10.1083/jcb.119.3.605 · Cited by 147 · see citing works
In Situ Detection of β-Galactosidase in Lenses of Transgenic Mice with a γ-Crystallin/ lacZ Gene
Goring, D. R., Rossant, J., Clapoff, S., et al. (1987) · Science · doi:10.1126/science.3099390 · Cited by 137 · see citing works
Transgenic mice demonstrate a testis-specific promoter for angiotensin-converting enzyme
Langford, K. G., Shai, S., Howard, T. E., et al. (1991) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(18)98437-1 · Cited by 129 · see citing works
Marker proteins for gene expression
Wood, K. V. (1995) · Current Opinion in Biotechnology · doi:10.1016/0958-1669(95)80009-3 · Cited by 116 · see citing works
Hurler's Syndrome: Deficiency of a Specific Beta Galactosidase Isoenzyme
Ho, M. W., & O’Brien, J. S. (1969) · Science · doi:10.1126/science.165.3893.611 · Cited by 106 · see citing works
Shimohama, S., Rosenberg, M. B., Fagan, A. M., Wolff, J. A., Short, M. P., Breakefield, X. O., Friedmann, T., & Gage, F. H. (1989) · Molecular Brain Research · doi:10.1016/0169-328x(89)90061-2 · Cited by 105 · see citing works
Indigogenic methods for glycosidases
Lojda, Z. K. (1970) · Histochemistry and Cell Biology · doi:10.1007/bf00306428 · Cited by 98 · see citing works
Young, D. C., Kingsley, S. D., Ryan, K. A., et al. (1993) · Analytical Biochemistry · doi:10.1006/abio.1993.1549 · Cited by 96 · see citing works
Day, E. D. (1981) · Springer eBooks · doi:10.1007/978-1-4684-3917-5_1 · Cited by 92 · see citing works
Reporter genes in transgenic mice
Cui, C., Wani, M. A., Wight, D. C., et al. (1994) · Transgenic Research · doi:10.1007/bf01973986 · Cited by 84 · see citing works
Specificity and multiple forms of β-galactosidase in the rat
Furth, A., & Robinson, D. (1965) · Biochemical Journal · doi:10.1042/bj0970059 · Cited by 82 · see citing works
Kapur, R. P., Hoyle, G. W., Mercer, E. H., Brinster, R. L., & Palmiter, R. D. (1991) · Neuron · doi:10.1016/0896-6273(91)90275-5 · Cited by 72 · see citing works
Indigogenic methods for glycosidases
Lojda, Z. K. (1971) · Histochemistry and Cell Biology · doi:10.1007/bf00264391 · Cited by 68 · see citing works
Login, G., Schnitt, S. J., & Dvořàk, A. M. (1987) · PubMed · Cited by 63 · see citing works
Weiss, D. J., Liggitt, D., & Clark, J. G. (1999) · The Histochemical Journal · doi:10.1023/a:1003642025421 · Cited by 55 · see citing works
Shaper, N. L., Harduin‐Lepers, A., & Shaper, J. H. (1994) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(17)31512-0 · Cited by 53 · see citing works
Article Commentary: The X-gal Caution in Neural Transplantation Studies
Sanchez‐Ramos, J., Song, S., Dailey, M., et al. (2000) · Cell Transplantation · doi:10.1177/096368970000900510 · Cited by 48 · see citing works
Tulsiani, D. R. P., Skudlarek, M. D., Araki, Y., et al. (1995) · Biochemical Journal · doi:10.1042/bj3050041 · Cited by 44 · see citing works
Chytil, F. (1965) · Biochemical and Biophysical Research Communications · doi:10.1016/0006-291x(65)90386-4 · Cited by 44 · see citing works
β- d -Glucosidases and related enzymic activities in pig kidney
Abrahams, H. E., & Robinson, D. (1969) · Biochemical Journal · doi:10.1042/bj1110749 · Cited by 40 · see citing works
Kolyada, A. Y., Lebedeva, T., Johns, C. A., & Madias, N. E. (1994) · Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression · doi:10.1016/0167-4781(94)90124-4 · Cited by 35 · see citing works
On Somatic Recombination in the Central Nervous System of Transgenic Mice
Abeliovich, A., Gerber, D., Tanaka, O., Katsuki, M., Graybiel, A. M., & Tonegawa, S. (1992) · Science · doi:10.1126/science.1631561 · Cited by 33 · see citing works
Rosenberg, W. S., Breakefield, X. O., DeAntonio, C., et al. (1992) · Molecular Brain Research · doi:10.1016/0169-328x(92)90240-c · Cited by 32 · see citing works
Spatial and temporal regulation of alacZ reporter transgene in a binary transgenic mouse system
Gardner, D. P., Byrne, G. W., Ruddle, F. H., et al. (1996) · Transgenic Research · doi:10.1007/bf01979920 · Cited by 30 · see citing works
Hara, Y., Nishimoto, J., & Suzuki, K. (1994) · Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression · doi:10.1016/0167-4781(94)90123-6 · Cited by 29 · see citing works
Nanba, E., & Suzuki, K. (1990) · Biochemical and Biophysical Research Communications · doi:10.1016/s0006-291x(05)81033-2 · Cited by 28 · see citing works
D'Agrosa, R. M., Hubbes, M., Zhang, S. M., et al. (1992) · Biochemical Journal · doi:10.1042/bj2850833 · Cited by 27 · see citing works
Human placental β-galactosidase. Characterization of the dimer and complex forms of the enzyme
Hubbes, M., D'Agrosa, R. M., & Callahan, J. W. (1992) · Biochemical Journal · doi:10.1042/bj2850827 · Cited by 25 · see citing works
Avé, P., Colucci‐Guyon, E., Babinet, C., et al. (1997) · Transgenic Research · doi:10.1023/a:1018401015497 · Cited by 19 · see citing works
Microwave-accelerated fixation and lacZ activity staining of testicular cells in transgenic mice
Murti, J. R., & Schimenti, J. C. (1991) · Analytical Biochemistry · doi:10.1016/0003-2697(91)90511-q · Cited by 18 · see citing works
Lazik, A., Liu, Y., Bringas, P., et al. (1996) · Trends in Genetics · doi:10.1016/0168-9525(96)99999-0 · Cited by 17 · see citing works
Hendrikx, P. J., Martens, A. C., Visser, J. W. M., & Hagenbeek, A. (1994) · Analytical Biochemistry · doi:10.1006/abio.1994.1516 · Cited by 12 · see citing works
Two gene products for beta-galactosidase are differentially expressed in the mouse salivary glands.
Nowroozi, N., Pa, D., Pc, D., et al. (1998) · PubMed · Cited by 7 · see citing works
Flagged first, then by citations
Cited by
Sorted by citations
Establishment and Maintenance of the Macrophage Niche
Guilliams, M., Thierry, G. R., Bonnardel, J., & Bajenoff, M. (2020) · Immunity · doi:10.1016/j.immuni.2020.02.015 · Cited by 512 · see citing works
Cai, Y., Zhou, H., Zhu, Y., Sun, Q., Ji, Y., Xue, A., Wang, Y., Chen, W., Yu, X., Wang, L., et al. (2020) · Cell Research · doi:10.1038/s41422-020-0314-9 · Cited by 397 · see citing works
Blast TBI models, neuropathology, and implications for seizure risk.
Kovacs, S. K., Leonessa, F., & Ling, G. S. F. (2014) · Frontiers in Neurology · doi:10.3389/fneur.2014.00047 · Cited by 71 · see citing works
Senescence-like phenotype in post-mitotic cells of mice entering middle age
Raffaele, M., Kovacovicova, K., Bonomini, F., Rezzani, R., Frohlich, J., & Vinciguerra, M. (2020) · Aging · doi:10.18632/aging.103637 · Cited by 28 · see citing works
- Authors
- Bolon, B.
- Affiliations
- Processus d'Activation Sélective par Transfert d'Énergie Uni-électronique ou Radiatif (FR)
- Corresponding author
- Bolon, Brad
- ORCID
- Bolon, Brad
- Venue
- Toxicologic Pathology
- Publisher
- SAGE Publications
- Published
- 2008-02
- Volume
- 36
- Issue
- 2
- Pages
- 265-276
- ISSN
- 0192-6233 (ISSN-L)
- 1533-1601
- OpenAlex
- W2130564466
- Cited by
- 23
- References
- 45
- Citations by year
- 2021: 3 · 2020: 4 · 2019: 1 · 2017: 2 · 2016: 1 · 2015: 4 · 2013: 1 · 2012: 3
- FWCI
- 0.76
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Toxicologic Pathology (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Animal Genetics and Reproduction
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Genetically Modified Organisms Research
- Keywords
- beta-galactosidase, lacZ transgene, pH-dependent enzyme activity, paraformaldehyde fixation, sex differences in enzyme activity
- MeSH terms
- Animals, Autopsy, beta-Galactosidase, Female, Histocytochemistry, Lac Operon, Lung, Male, Mice, Inbred C57BL, Mice, Transgenic, Staining and Labeling, Gene Expression, Rats, Sprague-Dawley, Mice, Rats
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
