False-Positive β-Galactosidase Staining in Osteoclasts by Endogenous Enzyme: Studies in Neonatal and Month-Old Wild-Type Mice

Abstract

Escherichia coli β-galactosidase (β-gal), encoded by the lacZ gene, has become an essential tool in studies of gene expression and function in higher eukaryotes. lac-Z is widely used as a marker gene to detect expression of transgenes or Cre recombinase driven by tissue-specific promoters. The timing and location of promoter activity is easily visualized in whole embryos or specific tissues using the cleavable, chromogenic substrate, 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-gal). The tissue specificity of promoters in transgenic constructs is routinely tested by using a promoter of choice to drive lacZ. Alternatively, the targeted expression of Cre recombinase to perform in vivo recombination of loxP sites can be visualized by β-gal staining in mice carrying a Cre-activated lacZ transgene, such as the ROSA26 strain. In the course of our investigations, we examined β-gal activity in bone tissue from genetically normal mice using standard detection methodology and found very high endogenous activity in bone-resorbing osteoclasts. This was true in frozen, paraffin, and glycol methacrylate sections. X-gal staining colocalized with the osteoclast marker, tartrate-resistant acid phosphatase (TRAP). β-gal activity was present in osteoclasts in long bones, in the mandible, and in both neonatal and more mature animals. We present this brief article as a caution to those testing genetic models of skeletal gene expression using β-gal as a marker gene.

Keywords:

• Gene Targeting
• Histochemistry
• LacZ
• Osteoclast Gene Expression
• Transgene Detection