Immunohistochemical staining of transgenic beta-galactosidase in burned skin is a better indicator of transfection efficiency than histochemical techniques.
The non-mammalian reporter gene LacZ, encoding the protein beta-galactosidase (beta-Gal), has long been used to test the efficiency of gene transfer into cells in culture or tissues in vivo. Biodistribution and dose-response transfection experiments rely upon a sensitive and specific technique for accurate results. We conducted an experiment to compare two techniques of identifying beta-galactosidase expression. The presence of beta-Gal was detected by dual staining transfected murine skin by both immunohistochemical (alkaline phosphatase) as well as histochemical staining (5-bromo-indolyl-beta-o-galactopyranoside [Bluo-Gal]). We demonstrated an almost two-fold increase in beta-galactosidase transfected dermal cell staining using immunohistochemistry as compared with histochemical staining. This translates to nearly 63% cells that were transfected with LacZ but were not identified by Bluo-Gal. The superior sensitivity of immunostaining suggests that anti-beta-Gal antibody represents the preferred analytical tool for light microscopic evaluation of LacZ gene transfer, promoter analysis in transgenic animals. Thus, identification of activity as opposed to presence of the enzyme underestimates gene expression following LacZ gene transfer in skin.
Pereira
Clifford
C
Shriners Hospitals for Children, Texas 77550, USA.
Maamar-Tayeb
Mokhtar
M
Burke
Ann
A
Perez-Polo
Regino
R
Herndon
David N
DN
Jeschke
Marc G
MG
D000818:Animals; D002057:Burns, Chemical / Q000378:metabolism*; D007150:Immunohistochemistry / Q000379:methods*; D008297:Male; D051379:Mice; D016612:Paraffin Embedding; D051381:Rats; D012867:Skin / Q000166:cytology / Q000378:metabolism*; D014162:Transfection; D001616:beta-Galactosidase / Q000378:metabolism*
D001616:beta-Galactosidase