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Mycoplasma Contamination Reduces the Effect of Lipid-Mediated Transfection,,,of Mammalian Cells


Mycoplasma PCR Primer Set for fast and easy detection of mycoplasma contamination

Leslie S. Arrington
Yale University

D. R. Staggs
Genetic Applications LLC

Heinz Miller
Stratagene

Mycoplasma contamination of mammalian cell cultures can substantially reduce the potency of transfection reagents.1 Cells must be free of mycoplasma contamination before the optimal amounts of LipoTAXI transfection reagent and DNA can be accurately determined. The use of Stratagenes Mycoplasma PCR Primer Set*, provides assurance that cells are free of mycoplasma contamination and able to maximally express transfected DNA.

The efficient introduction of foreign DNA into mammalian cells is vital to many areas of biological research. The ability to study the effect of introducing a gene or oligonucleotide of interest in a cell line may depend on the effectiveness of the transfection reagent. Stratagenes LipoTAXI transfection reagent can be used to achieve efficient transfections in a wide variety of mammalian cells. Using simple protocols, researchers can determine the optimal combination of LipoTAXI transfection reagent and plasmid DNA for a particular cell type.2 However, mycoplasma contamination can interfere with transfection using lipid, DEAE-dextran, CaPO4 and adenovirus-mediated methods.1 The resulting low transfection efficiencies may go undetected since antibiotic-resistant mycoplasma can exist in cell cultures without noticeable visual effects on the cells.3

In the typical protocol for optimizing transfection conditions, the amount of LipoTAXI reagent is increased at various concentrations of plasmid DNA that encodes for a reporter gene. Expression of the reporter gene increases until amounts of lipid and/or DNA prove toxic. While performing this optimization procedure in HeLa cells, we initially found unexpected results. The graph of our optimization study lacked the expected profile, and expression of the reporter molecule seemed unusually low and uncharacteristically flat (figure 1A).

figure 1

To determine whether mycoplasma was the cause of our atypical results, we used Stratagenes Mycoplasma PCR Primer Set to test the HeLa cells for contamination. This primer set provides a quick and sensitive method for assaying the presence of mycoplasma in cell cultures.4 We prepared cell-free supernatant samples according to the provided protocol and produced the PCR fingerprint shown in figure 2. The results in figure 2, lane 2 indicate that mycoplasma contamination was present in the cell culture at the time of the first optimization (figure 1A). The cells used in the first optimization study were destroyed, and the incubator and ventilated hood were carefully disinfected. Newly acquired cells were grown in the cleaned incubator and used for the second transfection. As shown in lane 3 of figure 2, mycoplasma contamination was not detected in the cells used in the second optimization study. In the second optimization study (figure 1B), increased detection of the b-galactosidase reporter molecule was seen. In this case, the data demonstrate the expected relationship between increasing amo unts of DNA and LipoTAXI transfection reagent found in optimization studies. Protein was measured in the cell lysates using a modified Bradford reagent and served as a measure of cytotoxicity. Based on the results shown in figure 1B and the protein data, 14 l of LipoTAXI transfection reagent and 1.5 g of DNA were selected as the conditions required in a 12-well plate format for optimal transfection of HeLa cells.

figure 2

Conclusions

Mycoplasma contamination of cell cultures occurs when an infected cell line is introduced to the laboratory.3 During optimization of transfection conditions using LipoTAXI reagent, low or atypical transfection results may indicate mycoplasma contamination. Stratagenes Mycoplasma PCR Primer Set includes internal controls and provides a quick test for mycoplasma. Researchers can use this primer set to detect contamination and regain high-efficiency transfection by eliminating mycoplasma contamination.

REFERENCES
  1. Xia, H., Fitzgerald, J., Bredt, D.S., and Forsayeth, J.R. (1997) Biotechniques 22: 934-936.

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

  3. McGarrity, G.J., and Kotani, H. (1985) In Mycoplasmas, Volume IV (S. Razin and M.F. Barille, eds.), pp. 353-386. Academic Press, New York.

  4. McKenzie, D., and Kaderli, M. (1993) Strategies 6: 58-59.

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


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