Histochemical Staining Following LacZ Gene Transfer Underestimates Transfection Efficiency

Abstract

Analysis of LacZ gene expression is conventionally inferred from blue staining that results from exposure of the transfected cells or tissue to the substrate 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal). Such histochemical staining reports not whether the gene product is present or absent, but where it is active. We investigated the hypothesis that identification of activity, as opposed to presence, of the enzyme underestimates gene expression following LacZ gene transfer. Under conditions optimized for in vitro histochemistry, up to 20% of cells stably transfected with nls-LacZ remained unstained by X-Gal. In contrast, immunostaining with a monoclonal or a polyclonal anti-β-galactosidase (β-Gal) antibody positively stained 99% of the cell nuclei. Following in vivo transfection of naked DNA encoding for nls-LacZ, X-Gal staining disclosed 2.7 ± 1.7 positive nuclei per LacZ-transfected animal, or a transfection efficiency of 0.015%. In contrast, immunohistochemical staining disclosed 118 ± 32.7 positive nuclei per transfected animal, yielding a transfection efficiency of 0.64% (p < 0.0001 versus X-Gal staining). Thus, 42.9 times more positive cells were detected by antibody than X-Gal staining. Finally, LacZ gene expression following intramuscular gene transfer with an adenoviral vector was observed in 7.6% of skeletal muscle cells assessed with X-Gal; anti-β-Gal antibody identified 21.8% of cells as being successfully transfected (p < 0.0001). Thus, X-Gal histochemistry following gene transfer of constructs encoding LacZ may underestimate the anatomic extent of gene expression. The superior sensitivity of immunostaining suggests that anti-β-Gal antibody represents the preferred analytical tool for light microscopic evaluation of LacZ gene transfer. Low transfection efficiency has been a consistent limitation of gene therapy. In many experimental studies, transfection efficiency has been evaluated using the reporter gene LacZ, encoding the protein β-galactosidase (β-Gal). Analysis of gene expression is conventionally inferred from β-Gal enzymatic activity upon exposure of transfected cells or tissue to X-Gal. We investigated the hypothesis that identification of activity, as opposed to presence, of the enzyme underestimates gene expression following LacZ gene transfer. X-Gal histochemistry following gene transfer of constructs encoding LacZ was found to underestimate the anatomic extent of gene expression assessed by immunostaining for β-Gal. The superior sensitivity of immunostaining suggests that anti-β-Gal antibody represents the preferred analytical tool for light microscopic evaluation of LacZ gene transfer.