Identification of transfected cell types following non-viral gene transfer to the murine lung.
BACKGROUND
Identification of the cell types transfected following gene transfer is an important factor in the selection of appropriate gene transfer agents (GTAs). Due to the relatively low gene expression mediated by non-viral GTAs, current methodologies for the detection and identification of transfected cells in the lung have proven insensitive and unreliable. We have investigated the use of the green fluorescent protein (GFP) to identify transfected cells in a mouse lung model.


METHODS
Direct visualisation of GFP fluorescence in frozen histological sections was used in conjunction with a panel of cell type specific antibodies to investigate the distribution and level of gene expression in mouse lungs following instillation of non-viral GTAs.


RESULTS
Despite considerable tissue autofluorescence, dose-dependent expression of GFP was detected following instillation of as little as 25 microg naked plasmid DNA (pDNA). Naked pDNA and pDNA complexed with polyethylenimine appeared to transfect mainly ciliated cells and Clara cells of the conducting airway, whereas expression mediated by pDNA complexed with the cationic lipid GL67 was found predominantly in type I pneumocytes.


CONCLUSIONS
Direct visualisation of GFP expression was used to detect transfected cell types in the mouse lung. In contrast with observations made using beta-galactosidase as a reporter, gene expression from several non-viral GTAs was readily demonstrated and no false GFP-positive cells were ever detected in untreated lung tissues. Lung delivery of different GTAs resulted in GFP expression in different cell types, confirming the importance of identification of transfected cells when screening and selecting GTAs for disease targets.
Davies
Lee A
LA
Gene Medicine Research Group, University of Oxford, John Radcliffe Hospital, Oxford, UK.
Seguela
Claire
C
Varathalingam
Anusha
A
Cheng
Seng H
SH
Hyde
Stephen C
SC
Gill
Deborah R
DR
D000818:Animals; D000906:Antibodies / Q000276:immunology; D005260:Female; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D008168:Lung / Q000737:chemistry*; D051379:Mice; D010957:Plasmids / Q000235:genetics; D014162:Transfection / Q000379:methods*
D000906:Antibodies; D049452:Green Fluorescent Proteins