PolyFect Transfection Reagent

Optimized for transfection of COS-7, NIH/3T3, HeLa, 293, and CHO cells

Features and benefits

• Optimized protocols for COS-7, NIH/3T3, HeLa, 293, and CHO cells
• Fast procedure and easy handling
• No removal of transfection complexes required
• Transfection in the presence of serum
• High efficiency
• Economical price

Principle

PolyFect Transfection Reagent is a solution of specifically designed activated-dendrimers. The reagent consists of dendrimer molecules of a defined spherical architecture with branches radiating from a central core (1; see figure "Activated-Dendrimer Structure"). The branches terminate at charged amino groups, which can interact with negatively charged phosphate groups of nucleic acids. PolyFect Reagent assembles DNA into compact structures (see figure "PolyFect–DNA Interaction"), that bind to the cell surface and are taken into the cell by nonspecific endocytosis. The reagent buffers the pH of the endosome, leading to pH inhibitition of endosomal nucleases, which ensures stability of PolyFect–DNA complexes.

Activated-Dendrimer Structure

Schematic representation of an activated dendrimer (1). Note the highly branched structure.

PolyFect–DNA Interaction

Model of the PolyFect–DNA complex. PolyFect Reagent (purple balls) interacts with DNA (black) to form a ring-like (toroid-like) structure. The upper right section of the illustration shows naked DNA, the lower section shows the interaction between dendrimers and DNA inside the complex, and the upper left section shows the final complete coverage of DNA within the complex.

Procedure

PolyFect Transfection Reagent is provided as a ready-to-use solution. The transfection procedure is fast and simple — just add PolyFect Reagent to the DNA solution, mix, incubate for 5–10 minutes, add growth medium (which can contain serum and antibiotics), and pipet the PolyFect–DNA complexes onto the cells. The cells are then incubated for gene expression. No post-transfection removal of complexes or medium change/addition is necessary, making the procedure fast and easy.

Applications

PolyFect Reagent has been specifically developed for fast and easy transfection of COS-7, NIH/3T3, HeLa, 293, and CHO cells. Optimized, cell-specific protocols for transfection of these cell lines in different cell-culture formats are provided in the PolyFect Transfection Reagent Handbook. In addition, optimized protocols for transfection of HeLa-S3 cells are available from QIAGEN Technical Services and the Transfection Tools web site (www.qiagen.com/transfectiontools/).

PolyFect Reagent consistently delivers high transfection efficiencies (see figures "High Transfection Efficiencies" and "PolyFect Reagent with HeLa Cells"). In contrast to many liposomal reagents, PolyFect Reagent enables transfection in the presence of serum without lowering of transfection efficiency.

High Transfection Efficiencies

Comparison of transfection efficiencies for PolyFect Reagent and two lipid-based reagents. Cells cultured in 6-well plates were transfected with a beta-galactosidase–reporter plasmid. The appropriate protocol was used for transfection with PolyFect Reagent. Optimized protocols were used for transfection with Reagents L2 and F; these were: 10 µl Reagent L2 with 3 µg DNA and 15 µl Reagent F with 2 µg DNA for CHO cells, and 10 µl Reagent L2 with 2 µg DNA and 7 µl Reagent F with 3 µg DNA for 293 cells. All transfections were performed in triplicate. Two independent transfection experiments are shown for 293 cells.

PolyFect Reagent with HeLa Cells

Expression of  A  β-galactosidase and  B  green fluorescent protein (GFP) in HeLa cells. Cells were cotransfected in 6-well plates with β-galactosidase– and GFP-reporter plasmids using PolyFect Transfection Reagent and the HeLa cell protocol. Expression was visualized by X-gal staining or fluorescence microscopy 2 days post-transfection.

High-throughput transfection

The application of recombinant DNA technology to fields such as drug discovery and development has led to an increased need for high-throughput transfection. Transfection using PolyFect Reagent requires minimal handling and removal of transfection complexes is not required, making this reagent highly suitable for high-throughput screening. PolyFect Reagent provides outstanding transfection efficiencies, excellent reproducibility, and minimal cytotoxicity in highthroughput transfection, and is available in bulk quantities. For optimized protocols for transfection of COS-7, NIH/3T3, HeLa, HeLa-S3, 293, and CHO cells in 96-well plates, please contact QIAGEN Technical Services or visit the Transfection Tools web site at www.qiagen.com/transfectiontools/.

操作手册:PolyFect Transfection Reagent Handbook 09/00 (PDF version, 163 KB)

Cited References

1. Tang, M.X., Redemann, C.T., and Szoka, Jr., F.C. (1996) In vitro gene delivery by degraded polyamidoamine dendrimers. Bioconjugate Chem. 7, 703.

For a complete list of references citing uses of QIAGEN products, visit the QIAGEN Reference Database online or contact QIAGEN Technical Services or your local distributor.