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High-Efficiency Transfections Achieved with New Low-Toxicity Reagent


Nonliposomal formulation yields high efficiencies in a variety of transfection conditions

Brenda Rogers Paul Kotturi Natalia Novoradovskaya
Stratagene

Introducing foreign DNA into eukaryotic cells requires high-potency transfections, which demand superior quality transfection reagents that are efficient, have low toxicity, and work in the presence or absence of serum. We compared Stratagenes new GeneJammer transfection reagent to each of five leading transfection reagents. GeneJammer reagent, a new nonliposomal formulation, not only performed well in numerous cell lines, it also yielded high efficiencies in primary cells. When the viability of transfected cells was evaluated, GeneJammer reagent showed the highest number of living transfectants for all cell lines tested.

The demand for high-efficiency transfections is increasing, reflecting the expanding applications for transfection technology. Introducing foreign DNA into eukaryotic cells has become fundamental to analyzing gene function, producing recombinant gene products, and devising strategies for gene therapy. Successful transfection results rely on the transfection method and the capability of the transfection reagent.1,2,3 The ideal transfection reagent is characterized by specific attributes, such as high efficiency in a wide variety of cells in the presence or absence of serum and the inclusion of antibiotics, growth factors, or other supplements. Other important attributes include low toxicity, high stability, and easy handling.

Stratagenes new GeneJammer transfection reagent, which is a proprietary formulation of a polyamine and other components, offers these essential qualitites for achieving successful transfection results. We subjected GeneJammer reagent and five of the most popular transfection reagents to extensive analyses, comparing transfection perform ance in protocols that show actual experimental conditions. In most of these comparative tests, GeneJammer reagent was more effective than the other transfection products; it showed high efficiencies in conditions that are typically problematic, such as in the presence of serum or in transfections of primary cells. Conveniently, GeneJammer reagent does not require reconstitution before use so handling time is minimized.

Optimizing Conditions

Before studies were compared, we optimized transfection conditions for GeneJammer and each of the five most common transfection reagents (products a, b, c, d, and e). We used the pCMV b-gal reporter plasmid (0.4 g/well) to transfect CHO-K1, COS-7, 293, HeLa, human umbilical arterial endothelial cells (HUAEC), and human umbilical vein endothelial cells (HUVEC) in 24-well plates. For optimization, we used four different concentrations of GeneJammer reagent (3, 6, 9, and 12 l per 1 g of DNA) and three concentrations of the competitive reagents within the ranges recommended by each manufacturer. Then the experiments were carried out according the manufacturers protocols.

Transfection assays with GeneJammer reagent included serum-free and serum-containing medium; the competitive reagents were used in the absence of serum. The in situ transfection efficiency was evaluated by the b-galactosidase histochemical staining assay; cell viability was calculated by the trypan blue exclusion assay. For each cell line, optimization was defined as the volume of each reagent that provided the highest transfection efficiency and the lowest toxicity. These optimized values were confirmed in three independent trials per cell line.

Comparing Transfection Efficiencies

Figure 1 shows the transfection efficiency of GeneJammer reagent in the presence and absence of serum, compared with the competitive rea gents. Both in fetal bovine serum (FBS) and in serum-free environment, GeneJammer-mediated transfection approached an efficiency of 90% to 100% in CHO-K1 and COS-7 cells . Adding FBS increased the amount of transfected HeLa and 293 cells from 38% and 50% to 65% and 92%, respectively. The efficacy of each competitive reagent varied among the different cell lines, with the following proportion of cells transfected: 50 to 95% of CHO-K1 cells, 30 to 80% of COS-7 cells, 10 to 22% of HeLa cells, and 25 to 85% of 293 cells. For all cell lines, transfections with GeneJammer reagent yielded satisfactory efficiencies. Twenty-four hours after transfection, we used Stratagenes in situ b-galactosidase staining kit to stain cells with X-gal, and the resulting cells that expressed for b-galactosidase were photographed (Figure 2).

Fig.2

Assessing Post-transfection Cell Viability

We evaluated the cytotoxic effects of GeneJammer and the competitive reagents by counting the cells stained with trypan blue . Briefly, 24 hours after transfections in the described conditions, media from each well was removed and saved. Cells were trypsinized, combined with saved media, pelleted, and resuspended in 25 l media. Aliquots were combined with an equal volume of 0.4% trypan blue stain. Cells were counted and the ratio of blue to white cells was determined. Of the cells transfected with GeneJammer reagent, 88% to 98% were viable 24 hours posttransfection, whereas the competitive reagents were associated with higher toxicity. The proportion of viable transfected cells was defined as a percent of transfected cells multiplied by the percent of viable cells (Figure 4). On all tested cell lines, GeneJammer reagent demonstrated a high transfection efficiency, low toxicity, and the highest number of viable transfectants.

Transfection of Primary Cells

One important measure of transfection reagents is how efficient they operate in cells that are resistant to transfection, such as primary cells. Typically, primary cells are less robust than transformed cells, yet the need for transfection of these types of cells is high. Successful transfection of primary cells allows studies in the more physiologically relevant biological models. Immortalized or transformed cell lines often have undefined phenotypes, and these phenotypes must be extensively characterized.

We transfected the pCMVbgal reporter plasmid into HUVEC and HUAEC cells, which are primary cells, with GeneJammer reagent or each of five competitors transfection reagents and compared the results. The best outcomes were obtained in serum-free media. The transfections were analyzed according to these parameters: transfection efficiency, total viable cells, and viable transfectants. Results (Figure 5) illustrate the high performance of GeneJammer reagent, confirming its capacity for efficient transfection in less-than-robust and difficult-to-transfect cells.

Conclusions

Use GeneJammer reagent, a polyamine proprietary formulation, to realize high transfection efficiency and low cytotoxicity when inserting DNA into cells. After comparing GeneJammer and five other leading transfection reagents, we found that GeneJammer reagent works well in the presence or absence of serum and, hence, is ideal to use when varying transfection conditions are desired. Moreover, GeneJammer reagent transfected the HUVEC and HUAEC primary cell lines with high efficiency, indicating its c apacity for high performance in many research applications, including transfection-resistant cells

REFERENCES

  1. Lasic, D.D. (1996) Adv. Drug. Del. 20: 221-266.

  2. Gao, X. and Huang, L. (1995) Gene Ther. 2: 710-722.

  3. Boussif, O., et al. (1995) Proc. Nat. Acad. Sci. USA 84: 7297-7301.


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