Identification of transfected cell types following non-viral gene transfer to the murine lung

• Published in: The journal of gene medicine 2007-03, Vol.9 (3), p.184-196
• Main Authors: Davies, Lee A., Seguela, Claire, Varathalingam, Anusha, Cheng, Seng H., Hyde, Stephen C., Gill, Deborah R.
• Format: Article
• Language: English
• Subjects: Animals; Antibodies - immunology; cystic fibrosis; Female; Gene therapy; Gene Transfer Techniques; GL67; green fluorescent protein; Green Fluorescent Proteins - analysis; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - immunology; lung; Lung - chemistry; Mice; non-viral gene transfer; Plasmids - genetics; Transfection - methods
• ISSN: 1099-498X; 1521-2254
• DOI: 10.1002/jgm.1007

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 µg 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 ß‐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. Copyright © 2007 John Wiley & Sons, Ltd.