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Low-Toxicity, Lipid-Mediated Transfection of Mammalian Cells

Optimizing transfection conditions using LipoTAXI transfection reagent

Peter L. Pingerelli
Stratagene Cloning Systems

Soverin Karmiol
Clonetics Corporation, Walkersville, Maryland

LipoTAXI transfection reagent is a novel liposome reagent that offers low cellular toxicity and high transfection efficiencies for adherent and suspended cells. A procedure is described for optimizing the amounts of LipoTAXI transfection reagent and DNA used to transfect various cell types. Data from the transfection of four human cell strains from the Clonetics Corporation illustrate typical optimization results.

Many facets of gene analysis and recombinant protein expression rely on continuous development and improvement of transfection technology, the procedures for transferring foreign DNA into mammalian cells.1,2 LipoTAXI transfection reagent offers improved, state-of-the-art transfections for diverse cell types and can be used to transfect both adherent cells and cells growing in suspension. Generally, the two most important parameters for achieving optimal transfection efficiencies are the concentration of DNA and the volume of LipoTAXI transfection reagent. By measuring the signal intensity of a reporter gene and the cellular proteins across a matrix of conditions, researchers can easily optimize the amounts of DNA and LipoTAXI transfection reagent for any cell type.

Optimizing Transfection Conditions

The control plasmid provided with LipoTAXI transfection reagent encodes the beta-galactosidase enzyme controlled by the cytomegalovirus (CMV) promoter. Varying amounts of the reporter plasmid were mixed with different volumes of LipoTAXI transfection reagent and added to cells plated in 12-well culture dishes. For these optimization studies, Clonetics Corporation provided the following cell types: small airway epithelial cell s (SAEC), prostate epithelial cells (PrEC), skeletal muscle cells (SkMC) and human mammary epithelial cells (HMEC). Forty-eight hours after transfection of these cell lines, relative expression of the reporter gene was compared3 to determine the optimal concentration of DNA and volume of LipoTAXI transfection reagent. In addition, the amount of protein in each well was measured using a modified Bradford protein reagent to assess the relative cytotoxicity for each set of transfection conditions. Decreasing protein content is an indication of cytotoxicity, whereas high protein content is associated with vigorous cell growth.

figure 1

The tradeoff between higher transfection efficiencies and increasing toxicity is shown in figures 1 and 2. In these experiments, 1 x 105 PrEC cells were plated in 12-well culture dishes and allowed to attach overnight. Reporter plasmid DNA and LipoTAXI transfection reagent were complexed together in serum-free media and then added to the cells. Cells were maintained at 37C in an unhumidified incubator with 5% C02. Signal production from the reporter gene was measured 48 hours following transfection using Stratagenes beta-Galactosidase Assay Kit (figure 1). These data show that an increase in the concentration of DNA results in greater signal production from the reporter gene. As the volume of LipoTAXI transfection reagent varied from 5 to 12.5 l and the amount of DNA varied from 0.5 to 1.5 g, the optimal quantities for each component were revealed. When cells were exposed to higher concentrations of DNA (figure 2), despite an improved signal from the reporter gene, cell protein levels decreased, an indication of incr eased cytotoxicity. Cytotoxic conditions result in cell death, which excludes a subpopulation of cells from transfection studies. In order to ensure a healthy, undamaged cell population and physiologically relevant transfection results, cytotoxic conditions should be avoided.

figure 2

LipoTAXI transfection reagent is consistently gentle to cells, as measured by the final concentration of cell protein per dish, even at high transfection efficiencies. However, the optimal amount of LipoTAXI transfection reagent and DNA varies for each cell line. Table 1 shows such variation in the described cell types.

Cell Type

DNA g

LipoTAXI reagent (l)

SAEC

1.0

5

PrEC

1.5

5

SkMC

1.5

5

HMEC

1.0

10

Table 1

Comparison to Other Commercially Available Transfection Reagents

The optimized amounts of DNA and LipoTAXI transfection reagent as defined in Table 1 were used for comparison to other commercially available transfection reagents. Each transfection reagent was used as recommended by the manufacturer. The best result for each reagent is reported in the graphs (figure 3, panels A, B, C and D).1 These results show LipoTAXI transfection reagent to be consistently effective when used for transfecting this diverse group of noncancerous, human cell lines. In addition, this reagent has been tested and shown to be effective in other cell types (data not shown).

figure 3

Comparison to Other Commercially Available Transfection Reagents

The optimized amounts of DNA and LipoTAXI transfection reagent as defined in Table 1 were used for comparison to other commercially available transfection reagents. Each transfection reagent was used as recommended by the manufacturer. The best result for each r eagent is reported in the graphs (figure 3, panels A, B, C and D).1 These results show LipoTAXI transfection reagent to be consistently effective when used for transfecting this diverse group of noncancerous, human cell lines. In addition, this reagent has been tested and shown to be effective in other cell types (data not shown).

Conclusions

LipoTAXI transfection reagent is a low-toxicity lipid formulation that provides high transfection efficiencies. This reagent consistently yields high transfection efficiencies in a variety of cell types and includes a reporter gene plasmid to be used for easy optimization of transfection conditions. Because LipoTAXI transfection reagent offers low toxicity, the integrity of transfected cells is maintained, yielding physiologically relevant transfection results.

REFERENCE

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

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

  3. Eustice, D.C., et al. (1991) Biotechniques 11: 739-743.


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