ExGen 500 in vitro Transfection Reagent (#R0511, 1ml (for in vitro transfections))

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1 1 ExGen 500 in vitro Transfection Reagent (#R0511, 1ml (for in vitro transfections)) Cationic polymer transfection reagent Concentration 5.47mM in nitrogen residues or 10µM in PEI. Reagent no Supplied 0.15M NaCl (endotoxin-free, sterile) solution for dilution of DNA. Formulation ExGen 500 is sterile apyrogenic solution of linear 22 kda polyethylenimine (PEI) in water. The solution is acidic and stable at 4 C, but its stability decreases if buffered. Description ExGen 500, polyethylenimine, belongs to an effi cient 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 effi ciencies when compared to various cationic lipids and polymers (1, 2, 3, 4). ExGen 500 has low toxicity and works effi ciently in the presence or absence of serum (1, 2, 3, 4, 5, 6). The effi cient 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 effi ciency of ExGen 500 is due to its protonation profi le, 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). 2 ExGen 500 and DNA charge-interact and form small (30-100nm), 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 effi ciency (10, 11, 12). Considerations for Transfection with ExGen DNA quality High quality DNA is critical for successful transfection. OD 260 / OD 280 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 (confl uency) for most cell types is 50-70% at the time of transfection. The cells should not be confl uent 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, β-galactosidase or Green Fluorescent Protein) that expresses an easily detectable protein. 4. Choice of promoter High transfection effi ciency depends on both the promoter under which the gene of interest is expressed and the cell

2 3 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 3nmol of phosphate and 1µl of ExGen 500 (10µM) is 5.47mM in nitrogen residues. µl of ExGen 500 x 5.47 No of equivalents = µg of DNA x 3 Initially, we recommend the use of 1µg of DNA and 3.3µl (6 equivalents) of ExGen 500 per well of 24-well plate (see Table 1). Subsequent optimization may further increase the transfection effi ciency in your particular application depending on the cell line and the gene expressed. The DNA quantity can range from 0.5 to 10µg 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 effi ciency is not affected by the presence of serum (2). In fact, the transfection effi ciency is higher in the presence of serum due to the better survival of cells Centrifugation Gentle centrifugation of tissue culture plates (for 5min at 280g) immediately after the addition of ExGen 500/DNA complex to the cells improves transfection effi ciency (14). I. 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).! Dilute 1µg of DNA in 100µl of 150mM NaCl. Vortex gently and spin down briefl y. " Add the 3.3µl ExGen 500 (not the reverse order) (11) and vortex-mix the solution immediately for 10sec. # Incubate for 10 minutes at room temperature. Notes. 1. Initially, we recommend the use of 1µg of DNA and 3.3µl (6 equivalents) of ExGen 500 per well of 24-well plate (see Table 1). 2. Subsequent optimization may further increase the transfection effi ciency in your particular application depending on the cell line and the gene expressed. % Add 100µl 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. & Gently rock the plate back and forth and from side to side to achieve even distribution of the complexes.

3 5 ' Centrifuge culture vessel, if possible, for 5min at 280g (14). ( 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. Table 1. Scale-up ratios were determined according to the surface area of the tissue culture vessel. Tissue Culture Vessel 96-well plate 48-well plate 24-well plate 12-well plate 6-well plate 35mm plate 60mm plate Growth Area (cm 2 /Well) Adherent cells to seed the day before transfection* x x x x x x x10 5 Amount of DNA (µg)** µl*** Volume of ExGen 500 (µl) at equivalents** II. Protocol for transfection of Jurkat cells! Use 1µg of DNA to transfect 1-2 x 10 5 Jurkat cells. Dilute intended quantity of DNA in 150mM NaCl to a fi nal concentration of 10µg/ml. Vortex gently and spin down briefl y. " Add 5.5µl of ExGen 500 for each 1µg of DNA (not the reverse order) (11) and vortex-mix the solution immediately for 10sec. # Incubate for minutes at room temperature. % Prepare a suspension of Jurkat cells (1-2 x 10 5 cells/ml) in complete growth medium. Cells should be at the logarithmic phase of growth with viability not less than 85%. & Add 100µl of the ExGen 500/DNA mixture to each 1ml of cell suspension. ' Gently mix and pipette into appropriate tissue culture vessel. ( Centrifuge culture vessel, if possible, for 5min at 280g (14). ) Incubate at 37 C for hours. * For transient transfection, monitor transgene expression. For stable transfection add the appropriate selective agent and monitor days after transfection. 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.

4 7 Troubleshooting Observation Possible cause Comments and Suggestions Transfection effi ciency is low. Transfection effi ciency varies in replicate experiments. Polyplex particles are not properly formed. The overall amount of polyplex particles is insuffi cient. The ExGen 500/DNA ratio is suboptimal. Incubation time is suboptimal for gene expression. The quality of DNA used is not high enough. Cell density was too high at the time of transfection. Inconsistent cell density in replicate transfections. 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. Modify the ExGen 500/DNA ratio. 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 (OD 260 /OD 280 greater than 1.8), free from RNA and oligonucleotides. Effective uptake of poliplex particles depends on the phase of cell growth and is most effective in logarithmic stage. Determine 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. 8 Observation Possible cause Comments and Suggestions Transfection effi ciency varies in replicate experiments. ExGen500 alone is toxic. ExGen500 alone is not toxic, but the ExGen500/ DNA complexes are toxic. Mycoplasma contamination Cell passage number is too high. The reagent contains endotoxin. The transfected gene or its product are toxic for the host cells. Excessive exposure to the polyplex particles. The overall amount of polyplex particles is too high. The ExGen 500/DNA ratio is suboptimal. Mycoplasma infected cells display signifi cant variations in their growth behavior and susceptibility to transfection that may cause low or/and inconsistent transfection effi ciency. 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 effi ciency of old cultures tends to decrease. Use cells with less than 50 splitting cycles. ExGen500 might be contaminated. Use another sample. Verify the transfected gene toxicity. 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. Modify the ExGen 500/DNA ratio.

5 9 References 1. Ferrari S., et al., Gene Ther, 4(10), , Bragonzi, A., et al., Gene Ther 6(12), , Ferrari S., et al., Biochim Biophys Acta, 1447(2-3), , Chemin I., et al., J Viral Hepat, 5(6), , Goula D., et al., Gene Ther, 7(6), , Zou S.M., et al., J Gene Med, 2(2), , Goula D., et al., Gene Ther, 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., Medecine/Sciences, 12, 56-59, Boussif O., 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 O., Zanta M.A., Behr J.P., Gene Ther, 3(12), , ExGen is a trademark of EUROMEDEX. 10 Cells successfully transfected with ExGen 500 include: Permanently growing cell lines 56FHT80 Human Fetal tracheal epithelium 6CFSMEo - Human Submucosal gland epithelium cells 9HTEo - Human Normal adult trachea epithelium cells A549 Human Type II pneumocytes B 16F10 Mouse Melanoma cells BNL CL.2 Mouse Hepatocytes C-26 Mouse Colon carcinoma cells C2C12 Mouse Myoblasts Caco-2 Human Colorectal adenocarcinoma cells CFNPE9o - Human Nasal epithelium cells CFPEo - Human Trachea epithelium cells Cos-7 Monkey African green monkey kidney cells CT26 Mouse Colon carcinoma cells HCS-2/8 Human Chondrocyte-like cells HeLa Human Cervix epitheloid carcinoma cells Hep 2C Human Epidermal carcinoma cells Hep G2 Human Hepatoma cells HL-60 Human Myelomonocytic cell line J-774 Mouse Myelomonocytic cell line Jurkat Human T cell leukemia KB Human Epithelial cells KBv Human Drug-resistant derivative of KB L929 Mouse Subcutaneous connective tissue fi broblasts LLC-MDR1 Porcine Drug-resistant derivative of LLC-PK1 LLC-PK1 Porcine Kidney epithelial cells MCA-38 Mouse Colon carcinoma cells MCF7 Human Breast adenocarcinoma cells MCF7 ADR Human Drug-resistant derivative of MCF7 Neuro2a Mouse Neuroblastoma cells NIH 3T3 Mouse Embrionic fi broblasts THP1 Human Myelomonocytic cell line U-937 Human Myelomonocytic cell line

6 11 Primary cell cultures of Tupaia belangeri Hepatocytes Pekin Duck Human, Rat Postmitotic neurons Human Monocytes / macrophages Newborn Rats Dorsal root ganglia neurons 12 Related Product ExGen 500 in vivo Transfection Reagent #R0521 Embrionic stem cells Human Quality Control Transfection efficiency was tested on COS-7 cells, using 1µg of pmok-mcgabgal plazmid and 4.3µl of ExGen 500 per 5 x 10 4 cells in 24-well plate. Transfection effi ciency, i.e. the percentage of transfected cells, is 91.0±1% as estimated by in situ X-gal staining. Quality authorized by: Margarita Leckiene PRODUCT USE LIMITATION. This product is developed, designed and sold exclusively for research purposes and in vitro use only. The product was not tested for use in diagnostics or for drug development, nor is it suitable for administration to humans. Please refer to for Material Safety Data Sheet of the product. 6 Revised