Detection of reporter gene expression in murine airways.
We have shown that to overcome the low levels of expression from gene transfer vector-mediated beta-galactosidase expression in lung, it is essential to replace the cytoplasmic beta-galactosidase gene with a nuclear targeted beta-galactosidase gene. We found that lung should be sectioned and fixed prior to staining for beta-galactosidase expression and that en bloc staining of intact lung is inefficient at staining positively transduced cells located deeper in the lung spaces. For GFP fluorescence, it is important to inflate the lungs with fixative prior to freezing and subsequent sectioning. For processing of nasal tissues for beta-galactosidase expression, we expand on a protocol used in previously reported gene transfer studies.
Limberis
Maria
M
Gene Therapy Program, Department of Pathology and Laboratory Medicine, University of Pennsylvania, USA.
Bell
Peter
P
Wilson
James M
JM
D000818:Animals; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D006651:Histocytochemistry; D008168:Lung / Q000378:metabolism*; D051379:Mice; D009297:Nasal Mucosa / Q000378:metabolism*; D013194:Staining and Labeling; D001616:beta-Galactosidase / Q000032:analysis* / Q000235:genetics / Q000378:metabolism
D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase