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Lipofection of Cultured Mouse Muscle Cells: a Direct Comparison of Lipofectamine and DOSPER

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Lipofection of Cultured Mouse Muscle Cells: a Direct Comparison of Lipofectamine and DOSPER Gene Therapy (1998) 5, 542–551 1998 Stockton Press All rights reserved 0969-7128/98 $12.00 http://www.stockton-press.co.uk/gt Lipofection of cultured mouse muscle cells: a direct comparison of Lipofectamine and DOSPER E Dodds1, MG Dunckley1, K Naujoks2, U Michaelis2 and G Dickson1 1Division of Biochemistry, School of Biological Sciences, Royal Holloway University of London, Egham, Surrey TW20 0EX, UK; and 2Division of Therapeutics, Boehringer Mannheim GmbH, Penzberg, Germany Cationic lipid–DNA complexes (lipoplexes) have been (GFP) were used in order to evaluate transfection widely used as gene transfer vectors which avoid the efficiency by histochemical staining or FACS analysis, adverse immunogenicity and potential for viraemia of viral respectively. Both lipid formulations were able to promote vectors. With the long-term aim of gene transfer into skel- efficient, reproducible gene transfer in C2C12 cells, and etal muscle in vivo, we describe a direct in vitro comparison to transfect primary mouse myoblast cultures successfully. of two commercially available cationic lipid formulations, However, DOSPER exhibited the important advantage of Lipofectamine and DOSPER. Optimisation of transfection being able to transfect cells in the presence of serum of was performed in the C2C12 mouse muscle cell line, both bovine and murine origin. This feature allowed before further studies in primary mouse myoblasts and increased cell survival during in vitro transfections, and C2C12 myotubes. Reporter gene constructs expressing may be advantageous for direct in vivo gene transfer either E. coli ␤-galactosidase or green fluorescent protein efficacy. Keywords: gene transfer; lipoplex; lipofection; skeletal muscle Introduction biosafety issues and adverse immune responses have led to the development of a range of alternative, nonviral Skeletal muscle provides an excellent target for the systems, where repeated administration is possible.4 expression of recombinant genes as its structure and Some of the most promising results have come from the function are well understood. It represents 30–50% of use of cationic lipids which are able to complex with total body mass and its easy accessibility lends itself well nucleic acid to form a lipoplex and deliver genes to the to the study of gene transfer techniques. A further advan- nucleus via the cellular endocytosis pathway, without tageous characteristic is the post-mitotic state of mature significant toxicity, immunology or germ cell localis- myofibres, which may facilitate long-term persistence of ation.5–7 The process of lipoplex formation involves elec- episomal transgenes. This feature is especially relevant to trostatic interactions between the cationic lipid and liposome-mediated gene transfer where stable inte- anionic DNA, as opposed to traditional liposome techno- gration of a transgene into the host genome does not nor- logies which result in encapsulation of DNA.8 Since their mally occur at a high frequency, so genetic material can introduction 10 years ago,9 many cationic lipid formu- be rapidly lost from transfected cells during repeated lations have been tested in a variety of in vitro situations rounds of cell division. Gene transfer into muscle has and have often yielded highly efficient transfection,10,11 been proposed for the treatment of inherited and superior to that obtained with encapsulating liposomes.12 acquired disorders both of muscle and nonmuscle tissues. However, studies administering lipoplexes to a range of For example, pre-clinical studies are currently underway tissues in vivo have led to mixed results. It seems that to try and alleviate the symptoms in animal models of cationic lipids may be suited to certain tissue types such primary muscle myopathies such as Duchenne muscular as lung or tumour13–15 where clinical trials are now in 1 dystrophy, using viral and nonviral techniques. A progress.16,17 Muscle presents a special case, where direct second major therapeutic application is to utilise muscle injection of naked plasmid DNA has resulted in signifi- tissues as an environment to express recombinant, non- cant levels of expression.18 By complexing the DNA with muscle proteins actively. This approach has been used cationic lipid, it may be possible to produce a more as a DNA vaccination system against, for example, the efficient and reproducible system of gene transfer into 2 hepatitis B virus, and as a means to augment levels of muscle, perhaps by promoting an alternative route of 3 serum proteins such as cytokines and lipoproteins. uptake. The concept of using lipoplexes as a method of High efficiency gene transfer into many eukaryotic DNA vaccination via intramuscular injection has already tissues and cell types has previously been achieved using been demonstrated.19 viral vectors. However, associated toxicity problems, As a prelude to direct in vivo gene transfer studies, we have compared the ability of two commercially available cationic lipids (Lipofectamine and DOSPER) to transfect Correspondence: G Dickson C2C12 cells, a mouse muscle cell line in vitro. Two differ- Received 8 August 1997; accepted 17 November 1997 ent reporter plasmids were utilised, first containing the Lipofection of muscle cells E Dodds et al 543 lacZ gene (pCMV␤), and second, the gene for green flu- transfection efficiency, all further studies were performed orescent protein (GFP; pUCUF2). Cultures were then using the lacZ reporter system and lipoplexes formed in assessed using histochemical staining or FACS analysis, these established optimal lipid:DNA ratios. respectively. Parameters were completely optimised in Lipoplexes formed in these optimal ratios were C2C12 cells, and then transfections into primary mouse additionally observed by negative staining TEM. Aggre- myoblasts and myotubes were performed using these gates of multilamellar vesicles were seen for both Lipo- optimal conditions, as these are likely to represent a fectamine–DNA and DOSPER–DNA lipoplexes (Figure closer model of the in vivo situation. In addition, formu- 3). The size of these complexes was extremely varied, lations suitable for in vivo injection were determined. with Lipofectamine–DNA complexes being significantly bigger than DOSPER–DNA complexes (100–830 nm and Ͻ Results 125–350 nm, respectively; P 0.05). Discrete lipid ves- icles approximately 100 nm in diameter were observed To establish maximal DNA transfection efficiency using in controls with lipid alone. the cationic lipids, a range of parameters were optimised. The optimisation studies were all performed in cultured Stability of expression C2C12 cells using either the lacZ or GFP reporter genes The stability of transgene expression was studied in a set in the plasmid expression vectors pCMV␤ and pUCUF2, of transfections in which wells were fixed and stained respectively. Direct comparison of transfection with the at varying time-points (20–72 h) following transfection. two vectors showed no significant difference in the opti- Maximal expression (25.6% ± 2.94 and 17.5% ± 2.32 for mal conditions found, indicating that the nature of the Lipofectamine and DOSPER, respectively; means ± s.d.) plasmid used is largely irrelevant. All experiments were was achieved by 18 h and remained at an equivalent level carried out in parallel for Lipofectamine and DOSPER up to 72 h (results not shown). These experiments in and each condition was repeated in triplicate in inde- C2C12 myoblasts could not be extended past 72 h as pendent cultures for statistical significance. Lipofectam- myoblast differentiation and fusion began to take place. ine-mediated transfections were performed in serum-free In all further studies expression was analysed 24 h medium, and DOSPER-mediated transfections in 5% FCS after transfection. medium unless stated otherwise. Time of incubation of complexes with cells Lipid:DNA ratio of complex An incubation time of 6 h is recommended by the manu- Lipoplex formation is dependent upon the charge ratio facturers for both DOSPER and Lipofectamine transfec- between the DNA and lipid.8 Therefore, a range of tions. This was re-examined in the myoblast system to weight to weight ratios was tested using the GFP reporter establish the effect of reduced incubation times on trans- system and FACS analysis (Figure 1). Transfections were fection efficiency and toxicity (Figure 4). It was found that performed in serum-free medium with Lipofectamine transfection efficiency increased up to 4 h (41.7% ± 3.5 for and 5% FCS medium with DOSPER. Cells were counted Lipofectamine and 37.0% ± 4.3 for DOSPER), and by FACS analysis 24 h after transfection. This revealed remained stable with longer incubation of the lipoplexes that a DOSPER:DNA ratio of 2:1 and a Lipofectami- with the cells up to 6 h. Nevertheless, an incubation time ne:DNA ratio of 7.5:1 led to maximal efficiency (Figure of 2 h may be preferable as significant efficiencies of 2). These are equivalent to charge ratios 30.5% ± 1.7 with Lipofectamine and 28.3% ± 2.4 with (positive:negative) of 2.73 and 11.2, respectively, showing DOSPER were achieved, with the advantage of increased that both lipoplexes have an overall positive charge (see levels of cell survival (56% ± 8.7 and 45.3% ± 9.3 for Lipo- Materials and methods). The amount of lipoplex required fectamine and DOSPER, respectively, at 2 h, as opposed for maximal transfection efficiency was detected by fixing to 17.7% ± 2.1 and 24.3% ± 4.6 at 4 h). As both Lipofect- the weight:weight ratio and altering the amount of DNA amine- and DOSPER-mediated transfections were perfor- and therefore also lipid added. For both Lipofectamine- med in serum-free medium for this set of experiments, and DOSPER-mediated transfections in 35 mm dishes, 4 and control cells exposed to serum-free medium for 6 h ␮g of DNA led to maximal efficiencies (33% ± 5.74 and showed notable levels of toxicity, the level of cell death 23% ± 0.68 of gated cells, respectively). Increasing the perceived is not entirely due to the transfection reagent, amount of DNA further had no significant increase on but in part to the incubation in serum-free medium.
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