( ...)Effectene Transfection Reagent

For transfection of a wide variety of cells, particularly effective for primary cells

Features and benefits

• Fast and easy transfection
  
• No transfection-complex removal needed for most cell lines
  
• Transfection in the presence of serum
  
• Ideal for primary cells or sensitive cell lines
  
• High transfection efficiencies and minimal cytotoxicity
• Low DNA requirement

Principle

Effectene Transfection Reagent is an innovative non-liposomal lipid formulation that is used in conjunction with a special DNA-condensing enhancer and optimized buffer to achieve high transfection efficiencies. The enhancer first condenses the DNA molecules and Effectene Reagent subsequently coats them with cationic lipids (see figure "Model of Effectene Principle"), providing a particularly efficient way of transferring DNA into eukaryotic cells. Effectene Reagent offers significant advantages over many liposome reagents and other transfection methods (1, 2).

Model of Effectene Principle

High Transfection Efficiencies Using Effectene Reagent
Comparison of transfection efficiencies using Effectene Reagent and two commonly used lipid-based reagents. Murine teratocarcinoma F9 cells (5 x 105) were transfected in 6-well plates with a luciferase-reporter plasmid using optimized conditions based on the manufacturer's instructions for each reagent. Transfection efficiencies were determined by measuring luciferase activity 48 h post-transfection, and are given as relative light units (RLU). (Data kindly provided by I. Clavereau, D. Petitprez, and I. Van Seuningen, Unit INSERM 377, Place de Verdun, Lille Cedex, France.)


Transfection of Oligonucleotides Using Effectene Reagent



C6 glioblastoma cells (1 x 104) were transfected with either naked FITC-labeled CD44 antisense oligodeoxynucleotide (ODN) or with EffecteneODN complexes. Intracellular accumulation and distribution of the transfected ODNs was analyzed 16 h post-transfection by immunofluoresence. A Naked ODNs (2 g). B ODNs (0.04 g) complexed with Effectene Reagent. Transfected ODNs are green in the photos. (Data kindly provided by G. Beutel and M. Schott, Institute of Neuropathology, Hanover Medical School, Hanover, Germany.)

Procedure

The Effectene procedure has two steps. DNA is first mixed with Enhancer and a buffer that provides optimal salt conditions for efficient DNA condensation. This step requires just 25 minutes. Effectene Reagent is then added and the mixture is incubated for 510 minutes to allow EffecteneDNA complexes to form. The complexes are mixed with growth medium (which can contain serum and antibiotics), and added directly to the cells. The cells are then incubated until harvested and analyzed for gene expression.

Applications

Effectene Reagent is suitable for transient and stable transfection of a broad range of cell types (see figures "High Transfection Efficiencies Using Effectene Reagent" and "Transfection of Oligonucleotides Using Effectene Reagent"). Because transfection can be performed in the presence of serum and requires low amounts of DNA (see figure "Serum and DNA Quantity vs. Transfection Efficiency"), cytotoxicity is minimal. This makes Effectene Reagent particularly effective for primary cells (see figure "Effectene Reagent with Primary Cells"). A searchable list of cell lines and primary cells successfully transfected using Effectene Reagent, as well as customer-developed transfection protocols, is available at the Transfection Tools web site www.qiagen.com/transfectiontools/.

Serum and DNA Quantity vs. Transfection Efficiency



Influence of serum and DNA quantity on transfection using Effectene Reagent. 2 x 104 COS-7 cells were seeded per well in 96-well plates one day before transfection. Cells were transfected using 0.011.0 g of a beta-galactosidasereporter plasmid and 0.088.0 l Enhancer (DNA: Enhancer ratio of 1:8) and 2 l Effectene Reagent, in either the presence or absence of serum. Each bar represents the average efficiency from four replicates 48 h post-transfection.

Effectene Reagent with Primary Cells

Expression of green fluorescent protein (GFP) in primary rabbit aortic smooth muscle cells transfected using Effectene Reagent. 1 x 105 cells were seeded one day prior to transfection, and transfections were performed in 6-well plates using 0.4 g of a GFP-reporter plasmid and 10 l Effectene Reagent per well. Cells were viewed 24 h post-transfection by fluorescence microscopy. Approximately 40% of the cells were transfected, as determined by FACS analysis. (Data kindly provided by K. Veit, 2nd Medical Clinic, Dept. Clinical Pharmacology, Mainz, Germany.)

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 Effectene Reagent requires low amounts of DNA and minimal handling, and in addition, removal of transfection complexes is not required, making this reagent highly suitable for high-throughput screening. Effectene Reagent provides outstanding transfection efficiencies, excellent reproducibility, and minimal cytotoxicity in high-throughput transfection, and is available in bulk quantities.

Cited References

1. Ausubel, F.M., et al. eds. (1991) Current Protocols in Molecular Biology, Wiley Interscience, New York.
2. Sambrook, J., Fritsch, E.F., and Maniatis, T., eds. (1989) Molecular cloning a laboratory manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.

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