Improved detection of lacZ reporter gene expression in transgenic epithelia by immunofluorescence microscopy.
The bacterial lacZ gene is commonly used as a reporter for the in vivo analysis of gene regulation in transgenic mice. However, several laboratories have reported poor detection of beta-galactosidase (the lacZ gene product) using histochemical techniques, particularly in skin. Here we report the difficulties we encountered in assessing lacZ expression in transgenic keratinocytes using classic X-gal histochemical protocols in tissues shown to express the transgene by mRNA in situ hybridization. We found that lacZ reporter gene expression could be reliably detected in frozen tissue sections by immunofluorescence analysis using a beta-galactosidase-specific antibody. Moreover, we were able to localize both transgene and endogenous gene products simultaneously using double-label immunofluorescence. Our results suggest that antibody detection of beta-galactosidase should be used to verify other assays of lacZ expression, particularly where low expression levels are suspected or patchy expression is observed.
Mahony
Donna
D
Department of Biochemistry and Molecular Biology and the Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD, Australia.
Karunaratne
Seetha
S
Rothnagel
Joseph A
JA
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D017930:Genes, Reporter / Q000502:physiology*; D006651:Histocytochemistry; D053553:Keratin-6; D015603:Keratinocytes / Q000502:physiology*; D007633:Keratins / Q000235:genetics; D007763:Lac Operon / Q000502:physiology*; D051379:Mice; D008822:Mice, Transgenic; D013194:Staining and Labeling; D014059:Tongue / Q000502:physiology; D001616:beta-Galactosidase / Q000276:immunology
D000906:Antibodies; D053553:Keratin-6; C507567:Krt6a protein, mouse; C507568:Krt71 protein, mouse; D007633:Keratins; D001616:beta-Galactosidase