Transfer of high copy number plasmid into mammalian cells by calcium phosphate transfection
Introduction
DNA-calcium-phosphate (Ca-Pi) coprecipitation is a commonly used method for gene transfer. Its usefulness in biomedical research and in biotechnological applications for the generation of highly productive recombinant cell lines has been largely demonstrated. However its application to gene therapy appeared promising only recently (Fasbender et al., 1998, Lee and Welsh, 1999, Walters et al., 2000). Despite its long history, qualitative and, particularly, quantitative studies on mechanisms of the Ca-Pi transfection method are rare. Using fluorescent 4′,6-diamidino-2-phenylindole dihydrochloride for plasmid labelling and fluorescent chlorotetracycline for calcium labelling in microscopic studies, Loyter et al. (1982) suggested that large quantities of plasmids are delivered into cells and remain concentrated in multiple ‘spots’, but only a very small proportion enters the nucleus. However, precise quantitative measurements were not possible, in part, because the labelling methods used resulted in reversible binding of fluorescent dye with plasmid DNA.
For the elucidation of intracellular distribution and degradation of plasmids during Ca-Pi transfection, Orrantia and Chang (1990) used radioactively labelled plasmids in studies utilizing subcellular fractionation: Less than 10% of plasmid was found in the nuclear fraction, remaining intact. In contrast, over 50% of intracellular radioactivity was associated with the cytosolic fraction, but represented completely degraded DNA. However the high density of Ca-Pi precipitates might interfere with fractionation and generate artefacts.
The covalent labelling of plasmids with fluorophores is an alternative approach to study plasmid uptake qualitatively and quantitatively. It avoids the drawback of reversible binding and it allows working on viable cells. Tseng et al., 1996, Tseng et al., 1997 used fluorescent ethidium monoazide covalently associated with plasmids by photoactivation for a study of transfection with cationic liposomes. In their study they used flow cytometry and demonstrated that cationic liposomes are able to deliver 105–106 copies of plasmid into the cells and that all cells with 106 or more copies were positive for Green Fluorescent Protein (GFP) expression. To obtain this result, they had to combine labelled and unlabelled plasmids because ethidium monoazide labelling strongly inhibits plasmid expression. More recently, Zelphati et al. (1999) developed another plasmid labelling method using fluorescent peptide nucleic acid (PNA). In this case, binding is not covalent but very strong and rather stable, due to the high affinity of PNA for specific DNA sequences (Zelphati et al., 1999). In addition, PNA labelling does not interfere with protein expression from the plasmid (Zelphati et al., 1999).
In our study on calcium phosphate transfection, we used both ethidium monoazide and PNA labelling and we analyzed plasmid uptake in individual cells. We developed quantitative flow cytometry to analyze the correlations between DNA uptake and other parameters known to influence transfection efficiency (exposure time to plasmid-Ca-Pi coprecipitates, method of Ca-Pi preparation, glycerol). We also investigated the role of endocytosis in Ca-Pi transfection, a mechanism of DNA uptake that was suggested by a preliminary study for Ca-Pi transfection (Loyter et al., 1982) and that was clearly demonstrated for liposomal transfection (Zelphati et al., 1999, Zabner et al., 1995).
Section snippets
Plasmids and labelled dextran
PNA labelled plasmids were obtained from Gene Therapy Systems (San Diego, USA). Plasmids used were either fluorescein labelled blank plasmid (5 kb, #G102000), rhodamine labelled GFP plasmid (6.5 kb, #G101040) or fluorescein labelled luciferase plasmid (6.7 kb, #G102035). Enhanced GFP (Clontech) was cloned into our own expression vector (6.9 kb), and was used for the experiments with ethidium monoazide. 2,000,000 Da, anionic and lysine-fixable dextran was from Molecular Probes (#D7137, Eugene,
PNA-fluorescein labelled plasmid DNA gives higher specificity and sensitivity than ethidium monoazide and PNA-rhodamine labelled plasmids
In order to compare the specificity and sensitivity of differently labelled plasmids, CHO cells were incubated either with PNA-fluorescein or with PNA-rhodamine labelled plasmids coprecipitated with Ca-Pi. Cells were analyzed subsequent to a 4 h incubation period and a brief glycerol treatment, according to an optimized protocol previously described (Jordan et al., 1996). Fluorescein and rhodamine labellings were analyzed by flow cytometry with a 488 nm (20 mW) and 514 nm (20 mW) excitation,
Acknowledgements
We thank Philippe Chatellard for his technical help.
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Present address: Ludwig Institute, Division d'Onco-Immunologie Clinique, Hopital Orthopedique, Niveau 5 - Aile Est, Avenue Paul Decker 4, 1005 Lausanne, Switzerland.