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Improve Lipid- or Calcium Phosphate-Mediated Transfection of Human Dermal,,,Fibroblasts


Improve transfection of primary cells up to 20-fold with Primary ENHANCER reagent

David Staggs
Genetic Applications LLC

Peter Pingerelli
Stratagene

Stratagenes Primary ENHANCER transfection reagent promotes efficient transfection of human dermal fibroblasts. Twenty minutes before transfection, cells are incubated in media containing the Primary ENHANCER reagent. After incubation, lipid or calcium phosphate (CaPO4)-mediated transfection can be performed using standard protocols. In earlier results, Primary ENHANCER reagent increased transfection efficiencies of some primary cell strains as high as 15-fold.1 In this study, we compared its effects on lipid- and CaPO4-mediated transfection using Stratagenes Normal Human Dermal Fibroblasts.

Human dermal fibroblasts are used to investigate many disease processes, including cancer, skin ulcers, collagen diseases, and scleroderma. They are also valuable for studying wound healing, growth factors, artificial skin, and dermal toxicity.

To study gene expression, a common approach usually involves introducing exogenous DNA into cells using transfection techniques in order to investigate the effects of resulting proteins. Unfortunately, primary human fibroblasts are difficult to transfect. However, with Stratagenes Human Dermal Fibroblasts, the method with which to study molecular and biochemical mechanisms in this system is improved. Additionally, Stratagenes new Primary ENHANCER reagent improves the transfection efficiency of lipid- or CaPO4-mediated transfection of human dermal fibroblasts.

Easily Increased Transfection

We grew Stratagenes Human Dermal Fibroblasts, which are isolated from adult skin and cryopreserved in vials (500,00 cells/vial),2 for two passages in standard growth media. The cells were then plated in 24-well format tissue culture dishes and allowed to attach for 18 hours. The media was replaced with media containing 0, 10, or 40 M of Stratagenes Primary ENHANCER reagent. After 20 minutes, the cells were transfected with a plasmid expressing the b-galactosidase reporter molecule under the control of the cytomegalovirus promoter (pCMVbGAL). Transfection was performed using either Stratagenes LipoTAXI transfection reagent3 or a CaPO4-mediated transfection technique.4

The Primary ENHANCER reagents effects on lipid-mediated transfection show that expression of the reporter molecule improved over 5-fold after incubating in 10 M Primary ENHANCER reagent and 20-fold after incubating in 40 M Primary ENHANCER reagent (Figure 1). Results were measured by Stratagenes fluorescent b-galactosidase assay kit.

Figure 1

The Primary ENHANCER reagents effects from using a CaPO4-mediated transfection technique show that expression of the reporter molecule, b-galactosidase, improved slightly after incubating in 10 M Primary ENHANCER reagent and nearly 3-fold after incubating in 40 M Primary ENHANCER reagent (Figure 2). Results were measured by Stratagenes fluorescent b-galactosidase assay kit.

Figure 2

Conclusions

Use Stratagenes Human Dermal Fibroblasts to study molecular and biochemical mechanisms in human fibroblasts. We demonstrated that the transient transfection of fibroblasts improved up to 20-fold in lipid-mediated transfection and nearly 3-fold in CaPO4-mediated transfection, using Stratagenes Primary ENHANCER reagent. When the need arises to transfect human fibroblasts, add the Primary ENHANCER reagent to a lipid-mediated or CaPO4-mediated transfection technique, and achieve optimal results.

REFERENCES

1. Halvorsen, Y.C., et al. (1998) Strategies 11: 58-60.
2. See package insert.
3. Petre, M., et al. (1998) Strategies 11: 88-90.
4. Ausubel F.M., et al., (1994) In Current Protocols in Molecular Biology. John Wiley & Sons, New York, NY.


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