( LipoTAXI reagent is effective w...)A Comparison Study of Lipid Tranfection Reagents in A549, NIH 3T3 and COS-7,,,Cell Lines

LipoTAXI reagent is effective with a wide variety of cells

Leslie S. Arrington
Genetic Applications LLC

LipoTAXI transfection reagent can be used for simple and reproducible delivery of foreign DNA into mammalian cells. In this study, we used varying amounts of LipoTAXI transfection reagent to optimize conditions for transfecting a reporter plasmid in three common cell lines. In addition, the efficacy of LipoTAXI reagent was compared to four other leading transfection reagents. In these studies, LipoTAXI reagent outperformed other transfection reagents and was less toxic to cells.

Researchers can improve the efficiency of introducing foreign DNA into different mammalian cell lines by optimizing transfection reagents. Lipid-mediated transfection reagents surpass other types of transfection reagents because they are easy to use, gentle to cells and provide consistent reproducible results.^1,2 However, among cell types, lipid-mediated transfection reagents can differ in transfection efficiency and proper dosage. Therefore, the first step to achieving high transfection efficiencies is to optimize the combination of lipid and DNA for each cell line.³ In this study, we examine the transfection efficiency of optimized LipoTAXI transfection reagent in A549, COS-7 and NIH 3T3 cell lines and compare this reagent to four other popular transfection products.

Optimization

For the initial optimization step, the signal strength of a reporter gene is measured as the amount of lipid is varied. The reporter plasmid (1.0 g) encoding the b-galactosidase gene was transfected into COS-7 cells with volumes of LipoTAXI transfection reagent that ranged between 6 and 15 l. The data demonstrate (Figure 1) that the highest transfection efficiency, as measured by b-galactosidase activity, occurred using 9 l of LipoTAXI reagent. This approach typically indicates the best amount of a transfection reagent for a particular cell line. Since high-level expression of a gene of interest may lead to unacceptably high cytotoxicity, total protein should be compared in cell lysates to select the optimal volume of LipoTAXI transfection reagent for the cell line studied.³

Figure 1

Comparison of Transfection Reagents

Using the results of the optimization experiment, we compared the efficacy of LipoTAXI transfection reagent to four other transfection reagents. Three of the reagents were cationic lipids; the fourth reagent was a branched polycationic polymer. All transfections included 1.0 g of reporter plasmid DNA. As determined for each cell line (Figure 1), the optimal amount of LipoTAXI transfection reagent was used. The other reagents were used at the volume recommended by the manufacturer. To ensure a fair comparison study, we also used the other reagents at slightly higher and lower amounts than recommended.

Figure 2

Analysis

The optimal amount of LipoTAXI transfection reagent varied considerably between cell lines. In general, the amount of transfection reagent that can be used is limited by cytotoxic effects of either the lipid or the expressed plasmid protein. The greater the amount of lipid tolerated by the cell line, the more transfection is achieved. To maximize the amount of lipid, we compared the concentration of cell proteins after transfections (data not shown). The A549 cell line seemed extremely tolerant to LipoTAXI transfection reagent as determined by the measurement of the total protein for the various volumes used. Even for 20 l of LipoTAXI transfection reagent, the concentration of cell protein was constant. For the other transfection agents, toxicity to cells increased as their amounts increased. Similar results were found when COS-7 and NIH 3T3 cell lines were transfected using 12 and 18 l of LipoTAXI transfection reagent, respectively. As shown in Figure 2, for the three volumes used, the other reagents demonstrated lower transfection efficiencies in all three cell lines.

Conclusions

These transfection studies first optimize LipoTAXI transfection reagent and then compare its efficacy to four other popularly used transfection reagents. Our comparison shows the superior efficiency and low toxicity of LipoTAXI reagent, features that make it the best transfection reagent for transfections in A549, COS-7 and NIH 3T3 cells.

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