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S U P P O R T
 

Considerations for Transfection with ExGen 500

(1) DNA quality
High quality DNA is critical for successful transfection. OD260/OD280 ratio of 1.8 or greater is recommended. DNA should be sterile and free of any contaminant such as endotoxins.

(2) Cell density at transfection
The recommended cell density (confluency) for most cell types is 50-70% at the time of transfection. The cells should not be confluent or at stationary phase at the time of transfection. See Table 1 below for growth areas of tissue culture vessels.

Table 1. Scale-up ratios were determined according to the surface area of the tissue culture vessel

Tissue culture vessel Growth area,
cm2/well		 Adherent cells to seed the day before transfection* Amount of DNA Volume of ExGen 500 (µl) at equivalents**
µg** µl***  5 6 7 8
96-well plate 0.3 0.5-1.2 x 104 0.3 20 0.8 0.99 1.15 1.32
48-well plate 0.7 1-3 x 104 0.5 50 1.37 1.65 1.92 2.19
24-well plate 2 2-6 x 104 1 100 2.75 3.3 3.84 4.39
12-well plate 4 0.4-1.2 x 105 2 100 5.5 6.6 7.68 8.78
6-well plate 9.5 0.8-2.4 x 105 3 200 8.23 9.87 11.52 13.2
35 mm plate 8 0.8-2.4 x 105 3 200 8.23 9.87 11.52 13.2
60 mm plate 20 2-6.3 x 105 5 200 13.71 16.45 19.2 21.9

Note:
Peak expression was determined on 24-well plates (2, 3, 6).
* Actual value depends on cell type.
** Estimated starting amount and may require optimization.
***The volume of DNA solution should represent 1/10 of the total volume of the culture medium.

(3) Transfection incubation time
Detection of transient expression of genes takes place within 2-72 hours. The optimal post-transfection incubation time can be determined and monitored using a reporter gene (such as Luciferase, beta-galactosidase or Green Fluorescent Protein) that expresses an easily detectable protein.

(4) Choice of promoter
High transfection efficiency depends also on both the promoter under which the gene of interest is expressed and the cell line used. 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.

(5) ExGen 500/DNA ratio (N/P ratio)

(6) Transfection in the presence of serum
Previously it has been shown that transfection efficiency is significantly decreased by the addition of serum to the incubation medium. This is particularly true when cationic lipids are used as vectors for gene transfer (14). On the other hand, ExGen 500 - mediated high transfection efficiency is not affected by the presence of serum (2).

(7) Centrifugation
Gentle centrifugation of tissue culture plates (for 5 min at 280 g) immediately after the addition of ExGen 500/DNA complex to the cells can improve transfection efficiency (15).

Protocol for in vitro transfection of adherent cells in a 24-well plate

Reagents to be supplied by the user:
Sterile solution of 150 mM NaCl is required to dilute ExGen 500 and plasmid DNA.

Quantities and volumes should be scaled up according to the number of cells/wells to be transfected. (See Table 1 for scale-up ratios).

(A) Preparation of the Polyplex particles (ExGen 500/DNA complex)

Prepare immediately prior to transfection

  1. Dilute 1 µg of DNA in 100 µl of 150 mM NaCl. Vortex gently and spin down briefly.

  2. Add 3.3 µl of ExGen 500 (not the reverse order) (11) and vortex-mix the solution immediately for 10 sec.
  3. Incubate for 10 minutes at room temperature.

Note

(B) Transfection

  1. Add 100 µl of the ExGen 500/DNA mixture to each well.
    Generally, the volume of the ExGen 500/ DNA mixture represents 1/10 of the total volume of the culture medium.

  2. Gently rock the plate back and forth, and from side to side to achieve even distribution of the complexes.
  3. Centrifuge culture vessel, if possible, for 5 min at 280g (see Considerations for Transfection with ExGen 500, Part 7).
  4. Incubate at 37°C for 24-48 hours.
    The above incubation is designed for transfection without media change. If media change is preferred, incubate for 30 min (if centrifugation is possible) or for 3-4 hours (if centrifugation is not possible). Replace the transfection complex with fresh complete growth media. Incubate for 24-48 hours.
The transfected gene expression can be monitored 24-48 hours (transient expression) or 10-15 days (stable expression) after the transfection.

Note

Low transfection efficiency

Cellular toxicity

  1. If ExGen 500 alone is toxic, ExGen 500 has been contaminated.

  2. If ExGen 500 alone is not toxic, but the ExGen 500/DNA complex is toxic:
Protocol for in vitro transfection of SF9 cells

According to I. D. Ogay et al, 2006, Transfection of insect cell lines using polyethylenimine. Cytotechnology 51(2):89-98.

  1. Incubate a confluent 25-cm2 T-flask of Sf9 cells at 4°C for 20 min. Remove the medium and replace with 6 ml of fresh FBS/Grace's medium with added antibiotic-antimycotics. Suspend the cells by gently bumping the flask followed by flushing with medium from a pipet. Count the cells and dilute them in complete Grace's to a density of 7x104 cells/ml. Uniformity in the cell seeding density between experiments is critical for reproducibility of transfection efficiency results.
  2. Seed 1 ml of suspended cells to each well of a 24-well tissue culture plate and allow 10-15 min for cells to attach at 22°C.
  3. Dilute 1.0 µg (2.0 µl) of DNA into 100 µl of 150 mM NaCl, vortex gently and spin down briefly for 5-10 seconds.
  4. Place tubes with DNA into the solid thermoblock and incubate at 72°C for 5-10 min for DNA sterilization. Allow the DNA solution to cool down to room temperature.
  5. Add 3.3 µl of ExGen500 (6 equivalents) to each tube with the DNA solution (not the reverse order) and vortex-mix the solution immediately for 10 seconds. Spin down briefly and incubate at room temperature for 10 min for generation of DNA/polyethylenimine complexes.
  6. Add 100 µl of DNA/polyethylenimine complexes by dropping slowly and evenly onto the cells to distribute the complexes. Seal the tissue culture plate and gently rock it back and forth and from side to side to achieve even distribution of the complexes. Centrifuge the culture plate for 5 min at 280 g.
  7. Incubate the transfected cells at 26°C in a plastic sealed container with damp paper towel to maintain humidity for 120 hours.

Transfection of 3T3, HepG2 or A549 cells was performed in the absence or presence of 10% serum. Expression of pCLuc was measured in relative light units (RLU)/mg protein. ExGen 500 high transfection efficiency is not affected in the presence of serum (2).

Human airway epithelial cells were transfected with pCLuc (in triplicate) using optimal conditions suggested by the manufacturers' protocols. Cells were harvested 24 h post-transfection and Luciferase gene expression was measured in relative light units (RLU). ExGen 500 showed the highest transfection efficiency in lung epithelial cells in the presence of serum (2).

pCMV-Luc was complexed with ExGen 500 then treated with DNase I for the specified time. Complexes were then used to transfect 3T3 cells. Luciferase expression was assayed 24 h post-transfection. Experiments were done in triplicate and results were measured in relative light units (RLU)/mg protein (3).

pCMV-Luc was complexed with ExGen 500 or lipid-based transfection reagent using optimal conditions suggested by the manufacturers' protocols. The complexes were treated with DNase I for the specified time. Transfection was carried out on 3T3 in triplicate. Luciferase gene expression was measured in relative light units (RLU)/mg protein. ExGen 500 showed greater protection of DNA from DNase degradation (3).

Lung epithelial cells were transfected with pCMV-Luc (in triplicate) using optimal conditions suggested by the manufacturers' protocols. Cells were harvested 24 h post-transfection and Luciferase gene expression was measured in relative light units (RLU). ExGen 500 showed the highest transfection efficiency in lung epithelial cells (1).

Transfection of different cell lines with 2 µg of pCMV-Luc complexed with ExGen 500. Plate centrifugation was carried out for 5 min at 280g. Expression of pCMV-Luc was measured in relative light units (RLU)/mg protein (16).

References:

  1. 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):1100-1106, 1997.

  2. 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.
  3. 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):219-225, 1999.
  4. 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):369-375, 1998.
  5. 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.
  6. 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):128-134, 2000.
  7. 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):712-717, 1998.
  8. 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):12349-12354, 1996.
  9. 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.
  10. 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.
  11. 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):7297-72301, 1995.
  12. Behr J.P., Gene transfer with synthetic cationic amphiphiles: prospects for gene therapy, Bioconjug Chem, Sep-Oct;5(5):382-9, 1994.
  13. 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 U S A, Apr 27;96(9):5177-5181, 1999.
  14. Felgner P.L., Gadek T.R., Holm M., Roman R., Chan H.W., Wenz M., Northrop J.P., Ringold G.M., Danielsen M., Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure, Proc Natl Acad Sci U S A, Nov;84(21):7413-7417, 1987.
  15. Boussif O., Zanta M.A., Behr J.P., Optimized galenics improve in vitro gene transfer with cationic molecules up to 1000-fold, Gene Ther, Dec;3(12):1074-1080, 1996.
  16. Boussif O., Transfert de gènes medie par des polymères cationiques, Thèse de doctorat, ULP Strasbourg, Decembre, 1996.
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Updated gegužės 10, 2007 10:58