( Achieve s...)Highest Transfection Efficiency of an Endotoxin-Sensitive Mammalian Cell,,,Line

Achieve superior transfection efficiencies with StrataPrep midiprep kit

Scott Basehore Jeff Braman
Stratagene

Plasmid DNA was purified with Stratagenes StrataPrep EF plasmid midiprep kit, a kit based on anion exchange chromatography, magnetic beads, and cesium chloride density gradient centrifugation. Plasmid yields and purity (as judged by 260-nm to 280-nm optical density ratios) were comparable with the four methods. However, endotoxin levels of the StrataPrep kit- and ion-exchange-purified plasmid DNA were nearly 800 times lower than magnetic bead-purified DNA and three times lower than DNA purified by cesium chloride density gradient centrifugation. The StrataPrep-purified plasmid yielded the highest efficiency transfection of an endotoxin-sensitive cell line when compared to the other three methods.

The StrataPrep EF plasmid midiprep kit is a simple and convenient method for obtaining up to 350 g of high-purity plasmid DNA from a single midi-spin cup device.¹ Contaminating endotoxin may be easily removed by using the kits unique endotoxin removal buffer (ERB). Endotoxin, a common contaminant of plasmid DNA isolated from gram-negative E. coli hosts, is difficult to remove²; its presence in plasmid preparations lowers the transfection efficiency of endotoxin-sensitive cell lines^2,3 and complicates the interpretation of experiments designed to examine signal transduction pathways triggered by a measured addition of endotoxin.⁴ Stratagenes unique ERB contains a color indicator to monitor endotoxin removal, thereby facilitating the purification process. Following endotoxin removal, plasmid DNA is bound to a special glass fiber matrix in a StrataPrep Kit midi-spin cup. Contaminants are removed by adding wash solution to the spin cup, centrifuging the cup, and eluting the purified DNA from the matrix in low-ionic-strength buffer. The resulting purified plasmid DNA is ready for subsequent molecular biology applications.

Four different plasmid purification methods were analyzed. Stratagene's StrataPrep EF plasmid midiprep kit, Qiagen's QIAfilter Plasmid Maxi kit with additional Endotoxin Free Buffer set (AEC: anion exchange chromotography), Promega's Wizard PureFection Plasmid DNA Purification system (MB: magnetic beads), and cesium chloride density gradient centrifugation (CsCl).

Yields and Purity

pCMV b-gal plasmid DNA was isolated from Epicurian Coli XL1-Blue MRF E. coli cells with the StrataPrep kit. Yields of plasmid DNA ranged from 360 g using the MB method to 473 g for CsCl. Yields of the StrataPrep Kit and AEC-purified DNA were 392 g and 433 g, respectively, for an equivalent volume of bacterial culture (Table 1).

The 260-nm to 280-nm optical density (OD) ratios ranged between 1.8 and 1.9 for each plasmid preparation (Table 1), indicating a high level of purity.⁵ OD values at 240 nm were also measured to detect other contaminants such as EDTA⁶ and detergents. The 260-nm to 240-nm OD ratios ranged between 1.5 and 1.7, which indicated that the plasmid preparations were essentially devoid of these contaminants⁶ (Table 1).

Endotoxin units per microgram of plasmid DNA (EU/g) were measured (BioWhittaker, Inc.) using the Kinetic QCL assay. Endotoxin levels for the StrataPrep Kit and AEC-purified DNA were approximately 800 times lower than MB-purified DNA and 3 times lower than CsCl-purified plasmid DNA (Table 1).

Transfection of Endotoxin-Sensitive Cell Line

The transfection efficiency for each plasmid preparation was determined by exposing 7.5 x 10⁶ endotoxin-sensitive RAW 264.7 cells⁴ to calcium phosphate precipitates containing 15 g of StrataPrep kit, AEC-, MB-, and CsCl-purified pA3-3.4Luc (IL-1Ra promoter/luciferase reporter) plasmid. Luciferase expression in this plasmid is stimulated by endotoxin. Cells were exposed to plasmid-calcium-phosphate precipitates for 5 hours, shocked in glycerol for 90 seconds, and washed. Cells from each plasmid transfection were divided equally into a set of six plates and returned to the incubator.

Forty hours post-transfection, three plates of each plasmid preparation set were treated with an excess (1 g/ml) of E. coli lipopolysaccharide (LPS), while the remaining three plates of each set were not stimulated. After an additional 8 hours of incubation, LPS-stimulated and unstimulated cells for each plasmid preparation were harvested, lysed, and assayed for luciferase activity and total protein. Relative light units (RLU) were normalized to protein concentration and expressed as RLU/mg of protein.

The transfection efficiency of StrataPrep-purified DNA, measured as total RLU/mg of protein for stimulated cells, was 1.3-, 3.4-, and 2340-fold higher than stimulated cells transfected with CsCl, AEC, and MB DNA, respectively (Figure 1). The fold RLU increase over background (RLU LPS-stimulated divided by RLU LPS-unstimulated control) for StrataPrep Kit-, CsCl-, AEC-, and MB-purified plasmid was 29, 18, 12, and 0.16, respectively. The high induction ratio for StrataPrep DNA reflects both a high transfection efficiency (indicated by enhanced LPS stimulation compared with the other preps) and negligible degree of endotoxin contamination, evident from the low LPS-unstimulated background measurement.

Fig.1

Conclusions

Purification of plasmid DNA with the StrataPrep kit is simple to perform and generates high yields of pure DNA. Because endotoxin is effectively removed, high-efficiency transfection of endotoxin-sensitive mammalian cells is easily achieved.

REFERENCES

1. Basehore, S. and Braman, J. (1999) Strategies 12: 5-7.

2. Cotton, M., et al. (1994) Gene Therapy 1: 239-246.

3. Weber, M., et al. (1995) BioTechniques 19: 930-939.

4. Sweet, M. and Hume, D. (1996) J. Leukocyte Biol. 60: 8-26.

5. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.

6. Held, P. (1998) Pharmaceutical Lab. February: 4-6.