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Transfection Reagents

ExGen 500 in vivo Transfection Reagent

Product information

Features

• Successful transfection in many animals including mice, rats, rabbits, tadpoles and ducks.

Applications

• Gene delivery via intravenous, intraventricular, subcutaneous, tracheal and intraperitoneal injections.

Description

ExGen 500 is a sterile solution of linear polyethylenimine (PEI) in water. PEI is a polymer with high cationic-charge density, which effectively condenses DNA for highly efficient gene-delivery (1, 2).

The ExGen 500 in vivo Transfection Reagent and DNA in a glucose solution form cationic microprecipitates which have a diameter of approximately 50 nm. The ExGen 500/DNA complexes interact with cell surface proteoglycans (syndecans) resulting in internalization by endosomes (1, 3).

Every third atom of the ExGen 500 backbone is a nitrogen atom of an amino group which binds protons. Therefore, the polymer acts as an effective proton sponge buffer within the endosomes (1, 4), protecting the internalized DNA from lysosomal degradation.

The ExGen 500 in vivo Transfection Reagent has been designed for in vivo transfection of any size DNA.

Concentration

The solution is 100 mM, in terms of nitrogen residues.

Storage

Store at 4°C.

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

General Considerations for Transfection

I. DNA Quality Requirements.
DNA quality is critical for successful transfection. Endotoxin-contaminated DNA may result in inefficient transfection and cause unacceptably high cellular toxicity. For DNA an A₂₆₀/A₂₈₀ ratio of 1.8, or greater is recommended.
II. Cell Density.
The recommended confluency for adherent cells on the day of transfection is 50-70% and 70-90% for TurboFect™ reagents. Suspension cells should be plated at an optimal density ensuring their logarithmic growth at the time of transfection.
III. Incubation Time.
Transient transgene expression takes place within 2-72 hours after DNA transfection. The optimal time depends on the cell type, promoter strength and expression product, and has to be determined experimentally. The recommended incubation time of cells with TurboFect™/protein complexes is 2 hours.
IV. Choice of Promoter.
High transfection efficiency depends both on the transgene promoter and on the cell line used. The cytomegalovirus (CMV) promoter is commonly used for high gene expression in a variety of cell lines. Other promoters, such as those from simian virus (SV40) and from Rous sarcoma virus (RSV) can also be used.
V. Transfection Reagent/Biomolecule Ratio.
The amount of transfection reagent used in transfection depends on the amount of DNA, siRNA or protein and cells to be transfected. The ratios presented in the protocols are starting ratios and can be further optimized for the best results.
VI. Transfection in the Presence of Serum.
Nucleic acid transfection efficiency using Fermentas transfection reagents is consistently high in the presence of serum. The presence of serum may reduce protein transfection efficiency by up to 50%. Therefore, protein transfection in serum-free medium is recommended for best results.
VII. Centrifugation.
Gentle centrifugation of tissue culture plates for 5 minutes at 280 x g after addition of the polyplexes can improve transfection efficiency.

In vivo DNA Transfection using ExGen 500 in vivo Transfection Reagent

Reagents to be Supplied by the User: sterile solution of 5% glucose (w/v) to dilute ExGen 500 and DNA.

1. Dilute 10 µg of DNA in 50 µl of a sterile 5% glucose solution. Vortex gently and centrifuge briefly.
2. Dilute 1.8 µl of ExGen 500 solution (6 equivalents) in 50 µl of sterile 5% glucose solution. Vortex gently and centrifuge briefly.
3. Add the diluted ExGen 500 to the diluted DNA (in this order). Vortex the solution immediately and spin down briefly.
4. Incubate for 10 minutes at room temperature.
5. Perform injections.
6. Monitor gene expression with the method most suitable for your studies.

Note

• The A₂₆₀/A₂₈₀ ratio should be at least 1.8 for purified DNA. It is important to use endotoxin-free DNA (less than 0.1EU/1 µg DNA).
• The amount of DNA and maximum injection volume depend on the experimental animal and the route of administration (see Tables 1 and 2 below) as well as on the targeted tissue or organ and on the expression vector.
• To prevent precipitation of the ExGen 500/DNA complex, the final concentration of DNA in the injection mix should not exceed 0.5 µg/µl.

Table 1. Suggested amount of DNA and maximum injection volume.

Animal	Route of injection	Suggested amount of DNA, µg	Maximum injection volume, µl	Reference
Adult mouse	intravenous injection	25-125	400-600	1, 6, 7, 9
	brain injection	2.5	5	5
Newborn mouse	brain injection	1	2	5
Nude mouse	intravenous injection	50	200	8
	subcutaneous tumor injection	10	100	8
Adult rabbit	tracheal injection	300-700	300-700	2, 4
Newborn rabbit	tracheal injection	300	300	4
Adult rat	brain injection	0.5	2	12
Tadpole	brain injection	0.5-1	1	10
Pekin Duck*	intravenous injection	400**	2000	3

Note

* 10 day old.
** 400 µg of fluorescein-labeled antisense oligodeoxynucleotides.
Table 2. Scale-up ratios

Amount of DNA, µg	Volume of ExGen 500 (µl) at different equivalents
	3	4	5	6	7	8	9
1	0.09	0.12	0.15	0.18	0.21	0.24	0.27
5	0.45	0.6	0.75	0.9	1.05	1.2	1.35
10	0.9	1.2	1.5	1.8	2.1	2.4	2.7
50	4.5	6	7.5	9	10.5	12	13.5

References

1. Bragonzi, A., et al., Conese M., Comparison between cationic polymers and lipids in mediating systemic gene delivery to the lungs, Gene Ther., Dec, 6(12), 1995-2004,1999.
2. Ferrari, S., et al., Polyethylenimine shows properties of interest for cystic fibrosis gene therapy, Biochim Biophys Acta, Oct 28, 1447(2-3), 219-25, 1999.
3. Chemin, I., et al., Liver-directed gene transfer: a linear polyethlenimine derivative mediates highly efficient DNA delivery to primary hepatocytes in vitro and in vivo, J. Viral Hepat, Nov, 5(6), 369-75, 1998.
4. Ferrari, S., et al., ExGen 500 is an efficient vector for gene delivery to lung epithelial cells in vitro and in vivo, Gene Ther., Oct, 4(10), 1100-6, 1997.
5. Goula, D., et al., Size, diffusibility and transfection performance of linear PEI/DNA complexes in the mouse central nervous system, Gene Ther, May, 5(5), 712-7, 1998.
6. Goula, D., et al., Rapid crossing of the pulmonary endothelial barrier by polyethylenimine/DNA complexes, Gene Ther., Mar, 7(6), 499-504, 2000.
7. Goula, D., et al., Polyethylenimine-based intravenous delivery of transgenes to mouse lung, Gene Ther., Sep, 5(9), 1291-5, 1998.
8. Coll, J.L., et al., In vivo delivery to tumors of DNA complexed with linear polyethylenimine, Hum Gene Ther., Jul 1, 10(10), 1659-66, 1999.
9. Zou, S.M., et al., Systemic linear polyethylenimine (L-PEI)-mediated gene delivery in the mouse, J. Gene Med, Mar-Apr, 2(2), 128-34, 2000.
10. Ouatas, T., et al., T3-dependent physiological regulation of transcription in the Xenopus tadpole brain studied polyethylenimine based in vivo gene transfer, Int J Dev Biol., Nov;42(8), 1159-64, 1998.
11. Boussif, O., et al., A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine, Proc Natl Acad Sci U S A, Aug 1, 92(16), 7297-301, 1995.
12. Fabre, V., et al., Homeostatic regulation of serotonergic function by the serotonin transporter as revealed by nonviral gene transfer, J Neurosci., Jul 1, 20(13), 5065-75, 2000.

References

1. Godbey, W.T., et al., Poly(ethylenimine) and its role in gene delivery, Controlled Release, 60, 149-160, 1999.
2. Goula, D., et al., Size, diffusibility and transfection performance of linear PEI/DNA complexes in the mouse central nervous system, Gene Therapy, 5, 712-717, 1998.
3. Kopatz, I., et al., A model for non-viral gene delivery: through syndecan adhesion molecules and powered by actin, Gene Med., 6, 769-776, 2004.
4. Behr, J.P., L’éponge a protons: un moyen d’entrer dans une cellule auquel les virus n’ont pas pensé, Médicine/Sciences, 12, 56-59, 1996.