HISTOCHEMICAL STAINING FOLLOWING LACZ GENE-TRANSFER UNDERESTIMATES TRANSFECTION EFFICIENCY

Analysis of LacZ gene expression is conventionally inferred from blue staining that results from exposure of the transfected cells or tissue to the substrate -bromo-4-chloro-3-indolyl-beta-D-galactopyranoside ( X-Gal). Such histochemical staining reports not whether the gene produ ct is present or absent, but where it is active. We investigated the h ypothesis 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. I n contrast, immunostaining with a monoclonal or a polyclonal anti-beta -galactosidase (beta-Gal) antibody positively stained 99% of the cell nuclei. Following in vivo transfection of naked DNA encoding for nls-L acZ, X-Gal staining disclosed 2.7 +/- 1.7 positive nuclei per LacZ-tra nsfected animal, or a transfection efficiency of 0.015%. In contrast, immunohistochemical staining disclosed 118 +/- 32.7 positive nuclei pe r transfected animal, yielding a transfection efficiency of 0.64% (p < 0.0001 versus X-Gal staining). Thus, 42.9 times more positive cells w ere detected by antibody than X-Gal staining. Finally, LacZ gene expre ssion following intramuscular gene transfer with an adenoviral vector was observed in 7.6% of skeletal muscle cells assessed with X-Gal; ant i-beta-Gal antibody identified 21.8% of cells as being successfully tr ansfected (p < 0.0001). Thus, X-Gal histochemistry following gene tran sfer of constructs encoding LacZ may underestimate the anatomic extent of gene expression. The superior sensitivity of immunostaining sugges ts that anti-beta-Gal antibody represents the preferred analytical too l for light microscopic evaluation of LacZ gene transfer.