(1) DNA quality
High quality DNA is critical for successful transfection. OD260/ OD280ratio of 1.8 or greater is recommended. DNA should be sterile and free of any contaminant such as endotoxins.(2) Choice of promoter
High gene expression depends on both the promoter under which the gene of interest is expressed and the targeted tissue/organ. Cytomegalovirus (CMV) promoter is best known for high gene expression in a wide variety of cell lines. Some researchers also prefer simian virus (SV40) and Rous sarcoma virus (RSV) promoters.(3) ExGen 500/DNA ratio (N/P ratio)
The required amount of ExGen 500 depends on the amount of DNA and the number of equivalents needed.
- One equivalent represents the amount of ExGen 500 required to neutralize the negative charges of the DNA phosphate groups.
- One µg of DNA is 3 nlol of phosphate and 1 µl of ExGen 500 (200 µM) is 100 mM in nitrogen residues.
Number of equivalents = µl of ExGen 500 x 100 --------------------------- µg of DNA x 3 - Initially, we recommend the use of 6 equivalents (5, 10). See Table 1 below for scale-up ratios, e.g. 10 µg of DNA would require 1.8 µl of ExGen 500.
- Optimal ExGen 500/DNA ratio can range from 3 to 12 equivalents depending on the gene expressed and the targeted organ.
Table 1. Scale-up Ratios
Amount of DNA, µg Volume of ExGen 500 (µl) at 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 13.5 100 9 12 15 18 21 24 27 (4) Amount of DNA and Maximum Injection Volume
The amount of DNA and maximum injection volume depend on the experimental animal and the route of administration (see Table 2 below) as well as the targeted tissue or organ and the expression vector.
- To prevent precipitation of ExGen 500/DNA complex, the final concentration of DNA in the injection mix should not exceed 0.5 µg/µl.
Table 2. 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 17 Tadpole Brain injection 0.5-1 1 10 Pekin Duck* Intravenous injection 400** 2000 3 * 10 day old.
**400 µg of fluorescein-labeled antisense ODN (oligodeoxynucleotides) AS2).
Protocol
Reagents to be supplied by the user:
Sterile solution of 5% glucose (w/v) is required to dilute ExGen 500 and DNA.
- Dilute 10 µg of DNA in 50 µl of sterile solution of 5% glucose. Vortex gently and spin down briefly.
- Dilute 1.8 µl of ExGen 500 solution in 50 µl sterile solution of 5% glucose. Vortex gently and spin down briefly.
- Add the diluted 50 µl of ExGen 500 to the diluted 50 µl of DNA (not the reverse order) (14). Vortex-mix the solution immediately and spin down briefly.
- Incubate for 10 minutes at room temperature.
- Inject animals.
- Monitor gene expression with the method most suitable for your studies.
pCLuc was complexed with ExGen 500 or other gene-delivery reagents using the optimal conditions suggested by the manufacturers' protocols. Mice were sacrificed 24 h post-injection and Luciferase gene expression in various organs was measured in relative light units (RLU). ExGen 500 showed the highest efficiency in all organs (1).
One µg of pCMV-Luc was complexed with ExGen 500 (at the indicated N/P ratio) in 5% glucose solution and injected into the third brain ventricle of Xenopus Tadpole. Animals were sacrificed 24h post-injection. Expression of pCMV-Luc in CNS was greater at 6 equivalents (10).
Mice were injected through the tail vein with ExGen 500/pCMV-Luc complex in 5% glucose solution. Animals were sacrificed at the time indicated. Expression of pCMV-Luc in various tissues was measured in relative light units (RLU)/mg protein. ExGen 500 showed exceptional gene delivery to various tissues (7).
One µg of pCMV-Luc was complexed with ExGen 500 in 5% glucose solution and injected into the third brain ventricle of Xenopus Tadpole. Animals were sacrificed at the time indicated. Expression of pCMV-Luc in CNS was measured in relative light units (RLU)/brain. ExGen 500 showed exceptional gene delivery to the central nervous system over 8 days (10).
Bragonzi A., Boletta A., Biffi A., Muggia A., Sersale G., Cheng S.H., Bordignon C., Assael B.M., Conese M., Comparison between cationic polymers and lipids in mediating systemic gene delivery to the lungs, Gene Ther., Dec;6(12), 1995-2004,1999.
- Ferrari S., Pettenazzo A., Garbati N., Zacchello F., Behr J.P., Scarpa M., Polyethylenimine shows properties of interest for cystic fibrosis gene therapy, Biochim Biophys Acta, Oct 28;1447(2-3), 219-25, 1999.
- Chemin I., Moradpour D., Wieland S., Offensperger W.B., Walter E., Behr J.P., Blum H.E., 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.
- Ferrari S., Moro E., Pettenazzo A., Behr J.P., Zacchello F., Scarpa M., 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.
- Goula D., Remy J.S., Erbacher P., Wasowicz M., Levi G., Abdallah B., Demeneix B.A., Size, diffusibility and transfection performance of linear PEI/DNA complexes in the mouse central nervous system, Gene Ther, May;5(5), 712-7, 1998.
- Goula D., Becker N., Lemkine G.F., Normandie P., Rodrigues J., Mantero S., Levi G., Demeneix B.A ., Rapid crossing of the pulmonary endothelial barrier by polyethylenimine/DNA complexes, Gene Ther., Mar;7(6), 499-504, 2000.
- Goula D., Benoist C., Mantero S., Merlo G., Levi G., Demeneix B.A., Polyethylenimine-based intravenous delivery of transgenes to mouse lung, Gene Ther., Sep;5(9), 1291-5, 1998.
- Coll J.L., Chollet P., Brambilla E., Desplanques D., Behr J.P., Favrot M., In vivo delivery to tumors of DNA complexed with linear polyethylenimine, Hum Gene Ther., Jul 1;10(10), 1659-66, 1999.
- Zou S.M., Erbacher P., Remy J.S., Behr J.P., Systemic linear polyethylenimine (L-PEI)-mediated gene delivery in the mouse, J. Gene Med, Mar-Apr;2(2), 128-34, 2000.
- Ouatas T., Le Mevel S., Demeneix B.A., de Luze A., 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.
- Mislick K.A., Baldeschwieler J.D., Evidence for the role of proteoglycans in cation-mediated gene transfer, Proc Natl Acad Sci U S A,Oct 29;93(22), 12349-54, 1996.
- Demeneix B., Behr J., Boussif O., Zanta M.A., Abdallah B., Remy J., Gene transfer with lipospermines and polyethylenimines, Adv Drug Deliv Rev, Mar 2;30(1-3), 85-95, 1998.
- Behr J.P., L'eponge á protons: un moyen d'entrer dans une cellule auquel les virus n'ont pas pense, Medecine/Sciences 12, 56-59, 1996.
- Boussif O., Lezoualc'h F., Zanta M.A., Mergny M.D., Scherman D., Demeneix B., Behr J.P., 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.
- Behr J.P., Gene transfer with synthetic cationic amphiphiles: prospects for gene therapy, Bioconjug Chem, Sep-Oct;5(5), 382-9, 1994.
- Godbey W.T., Wu K.K., Mikos A.G., Tracking the intracellular path of poly(ethylenimine)/DNA complexes for gene delivery, Proc Natl Acad Sci USA, Apr 27;96(9), 5177-81, 1999.
- Fabre V., Boutrel B., Hanoun N., Lanfumey L., Fattaccini C.M., Demeneix B., Adrien J., Hamon M., Martres M.P., Homeostatic regulation of serotonergic function by the serotonin transporter as revealed by nonviral gene transfer, J Neurosci., Jul 1;20(13), 5065-75, 2000.
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