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Transfection Reagents

TurboFect™ in vivo Transfection Reagent

Product information

Features

• No detectable inflamatory response.
• Gene delivery via multiple routes of administration.
• Reproducible results.
• Functionally tested in mice and rats.

Applications

• DNA transfection.
• siRNA-DNA co-transfection.

Description

TurboFect™ in vivo Transfection Reagent is a sterile solution of a cationic polymer in water. The polymer forms compact, stable, positively charged complexes with DNA. These complexes protect DNA from degradation and facilitate gene delivery in vivo. Reagent causes no detectable inflamatory response and is suitable for DNA administration via various routes, including intravenous, intraperitoneal, intratumoral injection, etc.

Storage

Store at 4°C.

Figure 1. No inflamatory response after DNA delivery using TurboFect™ in vivo Transfection Reagent.
50 µg of plasmid DNA was delivered into female BALB/c mice via intravenous injection using TurboFect™ in vivo Transfection Reagent. The concentration of proinflamatory cytokine TNFalfa was determined by ELISA in blood serum samples taken at the indicated time points. LPS – lipopolysaccharides.

Figure 2. Comparison of Fermentas TurboFect™ in vivo Transfection Reagent with other in vivo transfection reagents.
50 µg of plasmid DNA coding for firefly luciferase was complexed with in vivo transfection reagents and delivered into female BALB/c mice via intravenous injection. Transfections were performed according to manufacturers’ recommendations. Firefly luciferase expression was determined in various organs 24 h after plasmid DNA delivery.

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 TurboFect™ in vivo Transfection Reagent

Reagents to be Supplied by the User: sterile solution of 5% glucose (w/v).
Protocol

1. Dilute 50 µg of DNA in 400 µl of a sterile 5% glucose solution. Vortex gently and centrifuge briefly.
2. Add 6 μl of TurboFect™ in vivo Transfection Reagent and mix the solution by pipetting.
3. Incubate for 15-20 min at room temperature.
4. Perform injections.
5. 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 below) as well as on the targeted tissue or organ and on the expression vector.

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 TurboFect™ in vivo Transfection Reagent, µl
	recommended	range
1	0.12	0.1-0.16
5	0.6	0.5-0.8
10	1.2	1-1.6
50	6	5-8

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.