ExGen 500 in vitro Transfection Reagent

Transcription

1 ExGen 500 in vitro Transfection Reagent Catalog No: Lot No: XXXXX Supplied as: liquid Stability: store at +4 C Background ExGen 500, polyethylenimine, belongs to an efficient new class of non-viral, non-liposomal gene delivery reagents. It is capable of transfecting a wide variety of cell types both in vivo and in vitro {see attached list). It provides superior transfection efficiencies when compared to various cationic lipids and polymers (1, 2, 3, 4). ExGen 500 has low toxicity and works efficiently in the presence or absence of serum (1, 2, 3, 4, 5, 6). The efficient gene transfer activity of ExGen 500 is related to its capacity for condensing DNA, interacting with anionic proteoglycans of the cell membrane (2, 5, 7, 8), protecting DNA (3) and inducing endosomal swelling and rupture before DNA degradation (9). Principle The remarkable transfection efficiency of ExGen 500 is due to its protonation profile, which increases from 20-45% between ph 7.0 and 5.0. Every third atom is an amino nitrogen that can be protonated, thus making the molecule a virtual proton sponge (10,11,12). ExGen 500 and DNA charge-interact and form small ( nm), stable, highly diffusible particles that settle on the cell surface by gravity. The ExGen 500/DNA complex is then absorbed into the cell by endocytosis (7, 13). The proton sponge effect of the complex buffers the acidic ph of the endosome. This facilitates rupture of the vesicles and release of the ExGen 500/DNA complex in the cytoplasm before lysosomal degradation of DNA occurs. The protected DNA is then translocated to the nucleus resulting in high transfection efficiency (10, 11, 12). Concentration 5.47 mm in nitrogen residues or 10 µm in PEI. Reagents not supplied 0.15 M NaCI (endotoxin-free, sterile) solution for dilution of DNA. Formulation ExGen 500 is sterile apyrogenic solution of linear 22 kda polyethyl-enimine (PEI) in water. The solution is acidic and stable at 4 C, but its stability decreases if buffered. Considerations for Transfection with ExGen DNA quality High quality DNA is critical for successful transfection. The OD260/OD280 ratio should be greater than 1.6. 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 for growth areas of tissue culture vessels. 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, [3-galactosidase or Green Fluorescent Protein) that expresses an easily detectable protein.

2 4. Choice of promoter High transfection efficiency depends 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 prefer simian virus (SV40) and Rous sarcoma virus (RSV) promoters. 5. 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 nmol of phosphate and 1 µl of ExGen 500 (10 µm) is 5.47 mm in nitrogen residues. No of equivalents = µl of ExGen 500 x 5.47 µg of DNA x 3 Initially, we recommend the use of 1 µg of DNA and 3.3 µl (6 equivalents) of ExGen 500 per well of 24-well plate (see Table 1). Subsequent optimization may further increase the transfection efficiency in your particular application depending on the cell line and the gene expressed. The DNA quantity can range from 0.5 to 10 µg for 100,000 cells; likewise the ExGen 500/DNA ratio can range from 5 to 10 equivalents. 6. Transfection in the presence of serum ExGen 500 -mediated high transfection efficiency is not affected by the presence of serum (2). In fact, the transfection efficiency is higher in the presence of serum due to the better survival of cells. 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 improves transfection efficiency (14). Protocol for transfection of adherent cells in a 24-well plate Quantities and volumes should be scaled up according to the number of cells or wells to be transfected. (See Table 1 for scale-up ratios). 1. Dilute 1 µg of DNA in 100 µl of 150 mm NaCI. Vortex gently and spin down briefly. 2. Add the 3.3 µl 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: Initially, we recommend the use of 1 µg of DNA and 3.3/JI (6 equivalents) of ExGen 500 per well of 24-well plate (see Table 1). Subsequent optimization may further increase the transfection efficiency in your particular application depending on the cell line and the gene expressed. 4. Add 100 µl of the ExGen 500/DNA mixture to each well. Note: Generally the volume of the ExGen 500/ DNA mixture represents 1/10 of the total volume of the culture medium. 5. Gently rock the plate back and forth and from side to side to achieve even distribution of the complexes. 6. Centrifuge culture vessel, if possible, for 5min at 280g (14). 7. Incubate at 37 C for hours. For transient transfection monitore transgene expression. For stable transfection add the appropriate selective agent and monitore days after transfection.

3 Table 1. Scale-up ratios were determined according to the surface area of the tissue culture vessel. Tissue Culture Vessel Growth Area (cm 2 /Well) Adherent cells to seed the day before transfection* Amount of DNA Volume of ExGen (ul) 500 at equivalents** (ug)** up*** well plate x well plate x well plate x well plate x well plate x mm plate x mm plate x * Actual value depends on cell type **Estimated starting amounts may require optimization *** The volume of DNA solution should represent 1/10 of the total volume of the culture medium ExGen is a registered trademark of EUROMEDEX. Troubleshooting Observation Possible cause Comments and Suggestions Transfection efficiency is low. Polyplex particles are not properly formed. The overall amount of polyplex particles is insufficient. It is imperative to: add ExGen500 to DNA and not vice versa, immediately vortex the DNA/ExGen500 mixture. If the cytotoxicity is acceptably low, increase the overall quantity of DNA without changing the ExGen 500/DNA ratio. The ExGen 500/DNA ratio is suboptimal. Modify the ExGen 500/DNA ratio. Incubation time is suboptimal for gene expression. The quality of DNA used is not high enough. The optimal length of incubation with complexes may vary for different cell types. Try to increase, if possible, the contact time of the ExGen500/DNA complexes with the cells. Use high quality DNA (OD260/OD280 greater than 1.8), free from RNA and oligonucleotides.

4 Transfection efficiency varies in replicate experiments. ExGen500 alone is toxic. ExGen500 alone is not toxic, but the ExGen500- DNA complexes are toxic. Cell density was too high at the time oftransfection. Inconsistent cell density in replicate transfections. Mycoplasma contamination Cell passage number is too high. The reagent contains endotoxin. The transfected geneor its product are toxic for the host cells. Excessive exposure to the polyplex particles. The overall amount of polyplex particles is too high. Effective uptake of poliplex particles depends on the phase of cell growth and is most effective in logarithmic stage. Dettermine empirically the actual culture density corresponding to the logarithmic growth phase in your particular case. Count the cells accurately. Always seed the same number of cells and keep consistent the time between seeding and transfection. Mycoplasma infected cells display significant variations in their growth behavior and susceptibility to transfection that may cause low or/and inconsistent transfection efficiency. Test your cells for contamination. Use only mycoplasma free cultures. Cells that have been extended in culture for long time often tend to change their growth characteristics. Transfection efficiency of old cultures tends to decrease. Use cells with less than 50 splitting cycles. ExGen500 might be contaminated. Use another sample. Verify the transfected genetoxicity. Try to reduce the incubation time of ExGen500/DNA complexes to 2-3 hours. Decrease the overall quantity of DNA without changing the ExGen 500/DNA ratio. The ExGen 500/DNA ratio is suboptimal. Modify the ExGen 500/DNA ratio. References: 1. Ferrari S., et al., GeneTher, 4(10), , Bragonzi, A., et al., GeneTher 6(12),1995-4, Ferrari S., et al., Biochim Biophys Acta, 1447(2-3), , Chemin I., et al., J Viral Hepat, 5(6), , Goula D., et al., GeneTher, 7(6), , Zou S.M., et al., J GeneMed, 2(2), , Goula D., et al., GeneTher, 5(5), , Mislick K. A., Baldeschwieler J.D., Proc Natl Acad Sci USA, 93(22), , Demeneix B., et al., Adv Drug Deliv Rev, 30(1-3), 85-95, Behr J.P., Médecine/Sciences, 12, 56-59, Boussif 0., et al., Proc Natl Acad Sci USA, 92(16), , Behr J.P., Bioconjug Chem, 5(5), , Godbey W.T., Wu K.K., Mikos A.G., Proc Natl Acad Sci USA, 96(9), , Boussif 0., Zanta M.A., Behr J.P., Gene Ther, 3(12), , 1996.

5 Cells successfully transfected with ExGen 500 include: Permanently growing cell lines 56FHT80 6CFSME0 9HTEo A549 B 16F10 BNL CL.2 C-26 C2C12 Caco-2 CFNPE90 CFPEo Cos-7 CT26 HCS-2/8 HeLa Hep 2C Hep G2 HL-60 J-774 Jurkat KB KBv L929 LLC-MDR1 LLC-PK1 MCA-38 MCF7 MCF7 ADR Neuro2a NIH 3T3 THP1 U-937 Monkey Porcine Porcine Fetal tracheal epithelium Submucosal gland epithelium cells Normal adult trachea epithelium cells Type II pneumocytes Melanoma cells Hepatocytes Myoblasts Colorectal adenocarcinoma cells Nasal epithelium cells Trachea epithelium cells African green monkey kidney cells Chondrocyte-like cells Cervix epitheloid carcinoma cells Epidermal carcinoma cells Hepatoma cells T-cell leukemia Epithelial cells Drug-resistant derivative of KB Subcutaneous connective tissue fibroblasts Drug-resistant derivative of LLC-PK1 Kidney epithelial cells Breast adenocarcinoma cells Drug-resistant derivative of MCF7 Neuroblastoma cells Embrionic fi broblasts Usage This product is offered by Biomol for research purposes only. Not for diagnostic purposes or human use. It may not be resold or used to manufacture commercial products without written approval of Biomol GmbH.