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Versatile Transfection Reagent Offers Low Toxicity and Consistent,,,Performance


LipoTAXI transfection reagent: A proven choice

Matt C. Petre
Stratagene

LipoTAXI transfection reagent offers improved, consistent transfections for both adherent cells and cells growing in suspension. In this article, we summarize the results of several experiments that characterize the effectiveness of LipoTAXI reagent. Optimizing transfection conditions is a critical, preliminary step to achieving high transfection efficiencies, a need supported by data showing that transfection efficiencies vary with different cell lines and different lipid-transfection reagents. In addition, we examine the effect of mycoplasma contamination of cell lines on transfection efficiency. Finally, we show that LipoTAXI reagent is superior by comparing it to another lipid-based transfection reagent. Together, these studies confirm the high efficiency and low toxicity of LipoTAXI transfection reagent.

Using transfection procedures to introduce foreign DNA into mammalian cells is integral to many research endeavors. Consequently, focused efforts are directed toward improving transfection technology. Lipid-mediated transfection reagents have improved the technology because they are easy to use, gentle to cells, and yield consistent results.1,2 However, even for lipid-based procedures, a variety of factors can influence transfection efficiency. These include DNA concentration,3 choice and volume of transfection reagent,4 choice of cell line,5 and presence of mycoplasma contamination6.

To achieve high transfection efficiencies, we advise researchers using Stratagenes LipoTAXI transfection reagent to experimentally determine the concentration of DNA and volume of LipoTAXI transfection reagent. To optimize transfection in any cell type, it is standard to measure the signal intensity of a reporter gene and cellular protein concentration across a matrix of conditions. Using this approach, we performed several comparison studies to assess transfection efficiency and cellular toxicity, then determined that LipoTAXI transfection reagent consistently outperformed other lipid formulations.3,5

Optimizing Transfection Conditions

To perform convenient optimization studies, researchers can use the control plasmid included with LipoTAXI transfection reagent. This plasmid encodes the b-galactosidase enzyme regulated by the cytomegalovirus (CMV) promoter. We used this plasmid DNA (0.5 to 1.5 g) and LipoTAXI reagent (5 to 12.5 l), complexed together in serum-free media, to transfect 1 x 105 prostate epithelial cells (PrEC) from Clonetics Corporation (Figure 1A). Transfection conditions, preparation of cell lysates, and measurement of b-galactosidase activity were performed as previously described.3 In these experiments, increased b-galactosidase activity resulted from using higher DNA concentrations. However, as the concentration of DNA increased, cellular protein levels decreased (Figure 1B), indicating cytotoxicity. Cell death, associated with cytotoxicity, excludes a subpopulation of cells from transfection studies and should be avoided for results to be considered physiologically relevant. High protein content, which is associated with vigorous cell growth, is preferred.

Table 1
Optimized Concentrations of DNA and LipoTAXi Reagent

Cell Type

DNA (mg)

LipoTAXI reagent (ml)

SAEC

1.0

5

PrEC

1.5

5

SkMC

1.5

5

HMEC

1.0

10

We included data from optimization studies in several cell lines to illustrate that the cell-line variability of optimization results when using LipoTAXI reagent and DNA.3 Table 1 summarizes the findings of optimization studies for additional cell types from Clonetics Corporation: small airway epithelial cells (SAEC), skeletal muscle cells (SkMC), and human mammary epithelial cells (HMEC). For each cell type, the optimal amount of DNA and volume of LipoTAXI transfection reagent is indicated.

The cell-line variability of transfection efficiency was also demonstrated in another study that compared LipoTAXI reagent to four other lipid transfection reagents in A549, COS-7, and NIH 3T3 cell lines (Figure 2).5 This study used the b-galactosidase control plasmid and varying amounts of lipid reagents for transfections.

Figure 2

The b-galactosidase activity of transfected cell lysates indicated transfection efficiency. To determine toxicity, for each volume of transfection reagent used in each cell line, we measured the concentration of cellular proteins (data not shown). LipoTAXI reagent gave the highest transfection efficiency and lowest toxicity in all three cell lines.

Transfection Efficiencies: Effect of Mycoplasma Contamination

When standardization studies are performed to determine optimized amounts of LipoTAXI transfection reagent and DNA, low efficiencies or atypical transfection profiles may be observed. This may be due to mycoplasma contaminating the cell lines, since antibiotic-resistant mycoplasma can exist in cell cultures without noticeable visual effects on cells.

Figure 3

In one laboratory, optimization of LipoTAXI reagent using the b-galactosidase reporter plasmid in HeLa cells showed uncharacteristically low efficiencies.6 Stratagenes Mycoplasma PCR Primer Set,*, which provides a quick, sensitive assay for detecting mycoplasma in cell cultures, was used to test the HeLa cells for contamination. Cell-free supernatant samples were prepared, then analyzed, according to the protocol provided with the primer set. Results indicated that mycoplasma contamination was present in the HeLa cells at the time atypical optimization profiles were observed (Figure 3, lane 2). After the contaminated cells were destroyed and the laboratory was rigorously disinfected, a new source of HeLa cells was used for another series of optimization studies. These cells showed the expected, standard optimization profile and showed no contamination, as detected by the Mycoplasma PCR Primer Set (Figure 3, lane 3).

Comparing Lipid-Based Formulations

In most cases, researchers desire to define transfection conditions that produce the highest possible number of transfected cells for data analysis. For this reason, researchers want to use the reagent that reliably yields the highest transfection efficiencies for their cell lines. Several studies confirm that efficiencies vary for different lipid-mediated transfection reagents among cell types.5

One research group used an experimental system consisting of transient transfections of the human adrenalcarcinoma cell line (SW13), with a plasmid encoding the enhanced version of green fluorescent protein (EGFP). Calcium phosphate transfections yielded low transfection efficiencies and a corresponding subcellular localization of exogenous protein. To resolve these problems, they compared two liposome-based formulations, LipofectAMINE and LipoTAXI transfection reagents.4

The pEGFP-C3 plasmid, which encodes the EGFP with expression driven by the CMV promoter, and either LipofectAMINE or LipoTAXI reagent were used to transfect SW13 cells. The cells (8 x 104) were plated in 24-well culture dishes and allowed to attach overnight to achieve 60% to 80% confluency. For each liposome-based product, the manufacturers recommendations were used to determine cell-culturing specifications and the appropriate range for reagent volume and concentration of the plasmid DNA. Following transfections, cells were fixed, coverslips were prepared, and fluorescent cells were counted.4

Figure 4

A quantitative analysis of transfection efficiencies is shown for LipofectAMINE reagent (Figure 4A) and LipoTAXI reagent (Figure 4B). For all tested conditions, LipoTAXI transfection reagent yielded higher transfection efficiencies of SW13 cells with the pEGFP-C3 plasmid. In addition, cell death, indicated by floating cells, occurred with greater frequency with increasing concentrations of LipofectAMINE reagent. Increased cell death was not seen with increasing concentrations of LipoTAXI reagent, again demonstrating LipoTAXIs low toxicity.

Conclusions

Optimizing transfection conditions is a cell-line dependent process and should be performed for each cell line studied. When comparing efficiencies using four different lipid reagents for transfections in A549, COS-7, and NIH 3T3 cells, Stratagenes LipoTAXI transfection reagent consistently gave the highest transfection efficiencies and the lowest cellular toxicity. Atypical optimization profiles and low transfection efficiencies can be caused by mycoplasma contamination of cell lines, which is easily detected with Stratagenes Mycoplasma PCR Primer Set. LipoTAXI transfection reagent is significantly more effective than LipofectAMINE reagent for transfecting enhanced green fluorescent protein into SW13 cells, as measured by transfection efficiency and cell death. Furthermore, LipoTAXI transfection reagent is effective over a broader range of conditions than LipofectAMINE reagent leading to less cell death. Therefore, LipoTAXI transfection reagent is superior for consistent, low-toxicity, high-efficiency transfections.

REFERENCES
  1. Ausubel, F.M., et al. (1994) In Current Protocols in Molecular Biology. Wiley & Sons, New York.

  2. Jordan, M., Schallhorn, A., and Wurn, F.M. (1996) Nucleic Acids Res. 24: 596-601.

  3. Pingerelli, P., Petre, M., and Karmiol, S. (1997) Strategies 10: 8-9.

  4. Lowrie, D.J. (1997) Strategies 10: 114-115.

  5. Arrington, L.S. (1998) Strategies 11: 10-11.

  6. Arrington, L.S., Staggs, D.R., and Miller, H. (1998) Strategies 10: 112-113.

* U.S. Patent No. 5,491,062.


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