The main focus in proteomic studies is on the identification of proteins in given biological samples. Proteins isolated and separated from a given sample (e.g., whole cell lysates, blood or tissue, protein complexes) by immunoprecipitation or affinity chromatography or two-dimensional elect ... more
Efficient Transfection of Primary Human Skeletal Myoblasts Using FuGENE® HD Transfection ReagentStefanie Grunwald* and Astrid Speer
Molecular Biology, Department of Life Science and Technology, Technical University of Applied Sciences Berlin, Germany
Transfection of cells is one of the main techniques used to influence gene expression. Most primary cells and human skeletal myoblasts (SkMC) in particular are very difficult to transfect, whereas for cell lines such as C2C12, many suitable transfection reagents and protocols are available. Few publications report successful transfection of human primary myoblasts using non-viral systems [1,2,3]. These methods include cationic lipids such as phosphonolipids, electroporation, and a combination of liposome and adenoviral associated proteins. But transfection efficiency is low and often is a compromise between toxicity of the reagents and transfection efficiency.
Especially for primary SkMCs of individuals with dystrophies or atrophies, where cells are restricted, a technique with high transfection efficiency but low toxicity is needed. Therefore, we tested the efficiency and toxicity of FuGENE® HD Transfection Reagent in primary SkMCs and compared it with other reagents and systems.
Materials and Methods
Vector DNA preparation
The plasmid pReceiver M03 (Genecopoeia) was purified under endotoxin-free conditions according to manufacturer’s instructions. The length of the plasmid including a cDNA insert was 7 kb.
Cell culture and transfection
Primary SkMCs (Muscle Tissue Culture Collection; Munich, Germany) were cultured using skeletal muscle cell growth medium with 5% FCS (PromoCell, Germany) and antibiotics.
For transfection, the cells were trypsinized and 4 x 105 cells were plated into 6-well plates. Contrary to the FuGENE® HD Transfection Reagent protocol, the transfection was performed directly after seeding. Different FuGENE® HD Transfection Reagent:DNA ratios as well as different volumes of the FuGENE® HD Transfection Reagent-DNA-complex solution were used, as suggested by the protocol (Table 1). For example, 2 µg of DNA were diluted in 100 µl serum and antibiotic-free medium (OptiMem, Invitrogen). This was followed by the addition of 3 µl FuGENE® HD Transfection Reagent (ratio 3:2) and an incubation time of 15 minutes. This mixture (100%) was added to the cells at once. For 200% and 400%, the applied volume was doubled and quadrupled, respectively. The cells were incubated for 24 hours until the medium was changed with an additional washing step using fresh medium.
For comparison, cells were transfected with transfection reagents E, F, and with the system A as described in the suppliers’ protocols
Analysis of transfection efficiency
After 72 hours, the cells were washed, trypsinized, and washed again with 1x PBS before the lysis buffer TRI PURE was added. DNA was isolated according to manufacturer’s instructions. Additionally, RNase H and the restriction enzyme Stu I, which cuts the plasmid once, were added to the isolated DNA.
Real-time-PCR analysis was performed to measure the amount of transfected vector DNA. Two primer pairs were used. The first primer pair detected the cDNA insert of the vector as well as two known endogenous pseudo-genes of the insert as found at www.pseudogene.org. Since the cells used in all experiments originated from the same donor, the whole genome and consequently the number of pseudo-genes were identical and disregarded. The second pair was used to amplify endogenous DNA of the insert at which the forward primer corresponds to the forward primer for cDNA insert, but the reverse primer was designed within the next intron DNA.
A fourfold dilution series of vector DNA with triplicates of five different copy numbers was performed. A calculated standard curve was achieved with an efficiency of 1.99. For all samples, as given in Table 1, two real-time PCR reactions were performed: the amplification of the vector insert cDNA and the detection of endogenous DNA to correct variations of the amount of DNA utilized in each reaction. The adjusted results of all samples were applied to the standard curve to retrieve the copy numbers of each experiment.
Results and Discussion
Figure 1 presents the results for FuGENE® HD Transfection Reagent, the reagents E, F, and the system A. The highest transfection efficiency (i.e., 2.4 x 1010 copies isolated from 4 x 105 primary SkMCs) was obtained using a FuGENE® HD Transfection Reagent:DNA ratio of 6:2 and 200% complex volume. Even though this value appears extremely high at first, such a copy number was published for several primary cells . The authors showed that injections of 103 plasmid DNA copies per cell resulted in only a faint eGFP fluorescence signal in 30–50% of the cells. For a bright signal, a plasmid DNA copy number of 105 per cell was required. The negative controls, treated with FuGENE® HD Transfection Reagent only, still displayed a weak signal in real-time PCR, which may be a result rather from the two known pseudo-genes of the insert than from vector DNA contamination.
The reagents E, F, and the system A reached only a very low percentage of detected vector copies compared with FuGENE® HD Transfection Reagent. The transfection reagent E shows the best efficiency of these three, but reaches only 1/25 of the highest FuGENE® HD Transfection Reagent result. Additionally, it revealed a significant toxicity in contrast to FuGENE® HD Transfection Reagent. The impression of toxicity was monitored by microscopy 24 hours after incubation of cells with the FuGENE® HD Transfection Reagent-DNA complex as well as the other reagents and systems. No toxicity was determined for volumes of 100% and 200%, but moderate toxicity for 400%, whereas 30–40% of the cells died during transfection with reagent E, F, and with the system A (data not shown).
Using FuGENE® HD Transfection Reagent, primary human SkMCs can be successfully transfected with minimal toxicity. These cells are resistant to standard transfection reagents and are sensitive to toxicity.
In conclusion, FuGENE® HD Transfection Reagent offers for the first time a simple, affordable, and less toxic transfection method for transferring a 7 kb plasmid vector into primary human SkMCs.
Our next intention is to test whether the superior results can be replicated with another vector DNA and also with cells from probands suffering from muscular dystrophy whose proliferation rates are often low, a fact which may also influence the transfection efficiency.
1. Pampinella F et al. (2002) Mol Ther 5:161–169
2. Espinos E et al. (2001) Neuromuscul Disord 4:341–349
3. Campeau P et al. (2001) Gene Ther 8:1387–1394
4. Schindelhauer D, Laner A (2002) Gene Ther 9:727–730
This work has been supported by an operating grant from the German Society of Muscle Diseased Persons and a studentship of the Hypatia program, Technical University of Applied Sciences Berlin, Germany.
This article was originally published in Biochemica 3/2007, pages 26-27. ©Springer Medizin Verlag 2007
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