jetpei Cationic polymer transfection reagent In vitro Transfection Protocol 101-05 0.5 ml (250 transfections in 24-well plates) 101-05N 0.5 ml 50ml of 150 mm NaCl (250 transfections in 24-well plates) 101-10 1 ml (500 transfections in 24-well plates) 101-10N 1 ml 50ml of 150 mm NaCl (500 transfections in 24-well plates) 101-40 4 x 1 ml (2000 transfections in 24-well plates) 101-40N 4 x 1 ml 4 x 50ml of 150 mm NaCl (2000 transfections in 24-well plates) Protocol for in vitro DNA oligonucleotides delivery can be downloaded from our internet site (www.polyplus-transfection.com) or requested from support@polyplus-transfection.com. Content 1 ml of jetpei transfection reagent is sufficient to perform ca. 250 to 500 transfections in 24-well plates or 100 to 200 transfections in 60-mm dishes. Formulation and Storage jetpei is provided as a 7.5 mm solution in sterile and apyrogenic water (expressed as concentration of nitrogen residues). jetpei is shipped at room temperature and should be store at 4 C upon arrival. jetpei is stable for 1 year at 4 C. Description jetpei is a powerful transfection reagent that ensures effective and reproducible transfection with low toxicity 1. jetpei is a linear polyethylenimine, synthesized and purified by PolyPlus-transfection. It provides superior in vitro transfection when compared to various cationic lipids and polymers 2. JetPEI was shown to deliver genes to various established cell lines as well as primary cells 1, 3, 4. jetpei compacts DNA into positively charged particles capable of interacting with anionic proteoglycans at the cell surface and entering cells by endocytosis 5. It possesses the unique property of acting as a "proton sponge" that buffers the endosomal ph and protects DNA from degradation. The continuous proton influx also induces endosome osmotic swelling and rupture which provides an escape mechanism for DNA particles to the cytoplasm 1, 6, 7. Quality control Functional analysis: every batch of jetpei is tested by transfection into HeLa cells. Typically, transfection with a firefly luciferase gene (under the control of CMV promoter) gives 10 9 RLU (relative light unit)/ mg of proteins which corresponds to ca. 100 ng of luciferase protein. Definition of N/P ratio Efficient cell entry requires cationic particles. The ionic balance of jetpei cations and DNA anions should thus be in favor of the cations. The N/P ratio is a measure of the ionic balance of the complexes. It refers to the number of nitrogen residues of jetpei per DNA phosphate. Approximately one in three nitrogen atom of PEI is a cation, so electroneutrality of jetpei /DNA complexes is reached for N/P = 2-3. In practice, the best transfection results are obtained for N/P = 5-10. jetpei is supplied as a 7.5 mm solution (expressed in nitrogen residues) and 1 µg of DNA contains 3 nmoles of anionic phosphate. The amount of jetpei solution to be mixed with DNA in order to obtain a desired N/P ratio is given in table 1 and can be calculated using the following formula: (µg of DNA x 3) x N/P ratio µl of jetpei to be used = 7.5 Table 1. Volumes of jetpei solution and amounts of DNA for various N/P ratios. DNA N/P = 3 N/P = 5 N/P = 8 N/P = 10 1 µg 1.2 2 3.2 4 2 µg 2.4 4 6.4 8 4 µg 4.8 8 12.8 16 6 µg 7.2 12 19.2 24 8 µg 9.6 16 25.6 32 10 µg 12 20 32 40 1/12 2/12
Transfection Protocols 1. 3. Preparation of the complexes and transfection procedure jetpei is a very potent transfection reagent that can be used for more than hundred established cell lines as well as primary cells 1, 3, 4. The list of cell lines successfully transfected with jetpei may be consulted on our website: www.polyplustransfection.com under the Technical Support heading. jetpei cell line database displays optimal efficiencies and specific transfection conditions when different from the standard conditions described in this protocol. 1.Transient transfection of adherent cells 1. 1. Reagent required A 150mM NaCl sterile solution is required to dilute jetpei and DNA. This solution is provided with references N 101-05N, 101-10N and 101-40N. 1. 2. Cell seeding For optimal transfection conditions with jetpei, the cells should be 50-60% confluent. Typically, for transfection in 24-well plates, 50 000 to 100 000 cells are seeded per well, 24 hours before transfection. For other culture formats, refer to table 2 for the recommended number of cells to seed the day before transfection. Table 2. Recommended number of cells to seed the day before transfection 96-well 48-well 24-well 12-well 6-well/ 35 mm 6 cm/flask 25 cm 2 10 cm/flask 75 cm 2 14 cm/flask 153 cm 2 Number of adherent cells to seed 10 000-17 000 25 000-50 000 50 000-100 000 80 000-200 000 200 000-400 000 400 000-800 000 1 000 000 2 000 000 2 x 10 6 5 x 10 6 3/12 Surface area per well or plate (cm 2 ) 0.3 1 1.9 3.8 9.4 28 78.5 153 medium per well or plate* 0.1 ml - 0.2 ml 0.25 ml - 0.5 ml 0.5 ml - 1 ml 1 ml - 2 ml 2 ml - 4 ml 5 ml - 10 ml 10 ml 20 ml 20 ml 40 ml * to increase efficiency, use the lower volume of medium. In this case, 2h post-transfection, add serum-containing medium to double the volume. We recommend using N/P = 5. Refer to table 1 for other N/P ratios. The following protocol is given for transfection in 24-well plates, refer to table 3 for transfection in other culture formats. Table 3. Complexes preparation for different cell culture formats DNA (µg) the NaCl dilution solution (µl) 4/12 jetpei reagent (µl) jetpei dilution solution (µl) Total volume of complexes per well 96-well 0.25 10 0.5 10 20 48-well 0.5 25 1 25 50 24-well 1 50 2 50 100 12-well 2 50 4 50 100 6-well / 35 mm 3 100 6 100 200 6 cm 5 250 10 250 500 10 cm 7-8 500 14-16 500 1000 14 cm 10-12 1000 20-24 1000 2000 The optimal transfection conditions for a majority of adherent cell lines, as well as a general starting point for optimisation are given in the standard protocol described below. Check table 4 for different conditions optimised in some specific adherent cell lines. For each well, dilute 1 µg of DNA into 50 µl of 150 mm NaCl (provided with For each well, dilute 2 µl of jetpei solution into 50 µl of 150 mm NaCl. Vortex gently and spin down briefly. Add the 50 µl jetpei solution to the 50 µl DNA solution all at once (important: do Incubate for 15 to 30 minutes at room temperature.
Add the 100 µl jetpei /DNA mixture drop-wise onto the serum containing medium in each well and homogenize the mixture by gently swirling the plate. Transfection experiments are usually stopped after 24 to 48 hours and reporter gene activity assessed. 2.Transient transfection of suspension cells 2. 1. Cells seeding For optimal transfection conditions with jetpei, seed the number of cells adapted to the culture vessel format according to table 5. Table 5. Recommended number of cells to seed. Table 4: Optimal transfection conditions for some specific adherent cell lines. Cell line jetpei N/P transfection DNA (µg) reagent (µl) medium (ml) A 549 1 2 5 0.5 CaCo2 1 2 5 0.5 CHO 1 1.2 3 1 BHK 21 1 2 5 0.5 HEK 293 1 2 5 0.5 NIH 3T3 1 2 5 0.5 96-well 48-well 24-well 12-well 6-well / 35 mm 6 cm / flask 25 cm 2 10 cm / flask 75 cm 2 Number of cells 2.10 4-5.10 4 5.10 4 10 5 10 5 2. 10 5 2. 10 5 5. 10 5 5. 10 5 2. 10 6 2. 10 6 5. 10 6 5. 10 6-10 7 medium per well or plate 0.2 ml 0.5 ml 0. 5 ml -1 ml 1-2 ml 2-4 ml 4 8 ml 8 ml 16 ml DNA 0.2-0.4 µg 0.5-1 µg 0.5-1 µg 1-2 µg 2-4 µg 4 8 µg 8 16 µg Final volume of transfection mixture (µl/ well) 20 50 100 100 200 250 500 2. 2. Preparation of the complexes and transfection procedure 1. 4. Factors affecting transfection efficiency In contrast to other transfection reagents, jetpei is not affected by the presence of serum during transfection. Therefore, the jetpei /DNA complexes can be added directly to the serum containing medium 8. Usually, transfection efficiencies can be improved by using smaller volumes of medium (the lower volume indicated in table 2) or/and by centrifugation of the culture plate (5 min at 280g at room temperature) 8. If cytotoxicity is observed, the transfection complexes can be removed after a 2-4 hour incubation period. In this case, aspirate the medium containing the complexes and replace it with fresh serum-containing medium. We recommend using N/P = 5. Refer to table 1 for other N/P ratios. The optimal conditions of transfection for a majority of suspension cell lines, as well as a general starting point for optimisation are given in the standard protocol described below. Check table 6 for different conditions optimised in some specific suspension cell lines. Table 6: Optimal transfection conditions for some specific suspension cell lines. Cell line jetpei N/P transfection DNA (µg) reagent (µl) medium (ml) Daudi 2 4 5 0.5 Jurkat 2 6.4 8 1 K 562 2 4 5 0.5 Molt 4 2 4 5 0.5 5/12 6/12
The following protocol is given for transfection in 6-well plates. For each well, dilute 2 4 µg of DNA into 100 µl of 150 mm NaCl (provided with For each well, dilute 4 8 µl of jetpei solution (see table 1) into 100 µl of 150 mm NaCl. Vortex gently and spin down briefly. Add the 100 µl jetpei solution to the 100 µl DNA solution all at once (important: do Incubate for 15 to 30 minutes at room temperature. Add the 200 µl jetpei /DNA mixture drop-wise onto the serum containing medium in each well, homogenize the mixture by gently swirling the plate. Incubate at 37 C and 5% CO 2 in a humidified atmosphere. Transfection experiments are usually stopped after 24 to 48 hours and gene activity assessed. Cells growing in suspension are collected by centrifugation at 400g and then resuspended in the desired medium or buffer. The following protocol is given for transfection in 24-well plates. For each well, dilute 0.5 1 µg of DNA into 50 µl of 150 mm NaCl (provided with For each well, dilute 1 2 µl of jetpei solution (see table 1) into 50 µl of 150 mm NaCl. Vortex gently and spin down briefly. Add the 50 µl jetpei solution to the 50 µl DNA solution all at once (important: do Incubate for 15 to 30 minutes at room temperature. Add the 100 µl jetpei /DNA mixture drop-wise onto the serum containing medium in each well and homogenize the mixture by gently swirling the plate. Incubate at 37 C and 5% CO2 in a humidified atmosphere. Transfection experiments are usually stopped after 24 to 48 hours and gene activity assessed. Cells growing in suspension are collected by centrifugation at 400g and then resuspended in the desired medium or buffer. 3. Fast Protocol This protocol is optimized as a one day, time-saving procedure avoiding cell plating before transfection. Immediately following trypsinization, cells are transfected with jetpei in the presence of serum and then plated. This protocol provides the same high efficiency of transfection as the standard transfection protocol and can be useful for HTS applications. Pre-coating of wells with collagen or fibronectin is recommended to ensure good cell spreading (for 24-well plates, add 10 µg of collagen or fibronectin per well; for 96-well plates, add 2.5-5 µg of collagen or fibronectin per well). Table 7. Recommended number of cells relative to the culture vessel format 96-well 48-well 24-well Number of cells 10 000 20 000 25 000 50 000 50 000 100 000 medium per well 200 µl 400 µl 500 µl DNA 0.1 0.2 µg 0.25 0.5 µg 0.5 1 µg Final volume of transfection mixture per well 50 µl 50 µl 100 µl 3. 1. Transfection in 24-well plates Preparation of the complexes We recommend using N/P = 3-5. Refer to table 1 for other N/P ratios. For each well, dilute 1 µg of DNA into 50 µl of 150 mm NaCl (provided with For each well, dilute 1.2-2 µl of jetpei solution into 50 µl of 150 mm NaCl. Vortex gently and spin down briefly. Add the 50 µl jetpei solution to the 50 µl DNA solution all at once (important: do Incubate for 15 minutes at room temperature. Preparation of cells and transfection After trypsinization, wash the cells once with fresh serum-containing medium, then seed 100 000 cells in 500 µl medium with serum in a sterile tube. Add the 100 µl jetpei /DNA mixture to each tube and immediately vortex-mix the mixture gently. 7/12 8/12
Transfer the cells/dna complexes solution into a well (pre-coated with collagen or fibronectin). Incubate at 37 C with 5% CO2. Transfection experiments are usually stopped after 24 to 48 hours and reporter gene activity assessed. 3. 2. HTS protocol (transfection in 96-well plates) Preparation of the complexes For each well, dilute 200 ng of DNA into 25 µl of 150 mm NaCl (provided with references N 101-05N, 101-10N and 101-40N). Vortex gently. For each well, dilute 0.4 µl of jetpei solution into 25 µl of 150 mm NaCl. Vortex gently. Add the 25 µl jetpei solution to the 25 µl DNA solution all at once (important: do Vortex-mix the solution immediately and spin down briefly. Incubate for 15 minutes at room temperature. Preparation of cells and transfection (for 96-well plates precoated with collagen or fibronectin). After trypsinization, wash the cells once with fresh serum-containing medium, then seed 20 000 cells in 200 µl medium with serum per well. Add the 50 µl jetpei /DNA mixture to each well and homogenize the mixture by gently swirling the plate. Incubate at 37 C with 5% CO2. Transfection experiments are usually stopped after 24 to 48 hours and reporter gene activity assessed. 4. Stable transfection For stable transfection, perform transfection in 6-well plates or 60 mm plates according to the protocol described in section 1. Start selection with appropriate antibiotic 24 48 h after transfection. A detailed protocol can be downloaded from our Internet site (www.polyplus-transfection.com). Troubleshooting Problems Low transfection efficiency Cellular toxicity Comments and Suggestions Optimize the amount of plasmid DNA used in the transfection assay. Use high-quality plasmid preparation, free of RNA (the OD260/280 ratio should be greater than 1.8). Ensure that adherent cells are 50-60% confluent the day of transfection. Optimize the jetpei /DNA ratio starting from 1µl jetpei /µg DNA up to 4µl jetpei /µg DNA. Perform a positive control transfection experiment with a well-characterized reporter gene (Luciferase or ß-Gal from commercially available plasmid). Decrease the culture medium volume. Gently centrifuge the culture plates (if the cells can withstand it), usually 5 min at 280g. Decrease the amount of plasmid DNA used in the transfection assay (keeping the jetpei /DNA ratio constant). Check DNA concentration and ensure that jetpei /DNA ratio is no more than 2µl of jetpei for 1 µg of DNA. Reduce the incubation time of the complexes jetpei /DNA with the cells. Verify the toxicity of the expressed protein. If the expressed protein is toxic for the cells, reduce the amount of plasmid DNA used in the transfection assay. Make sure that the plasmid preparation is endotoxins-free. 9/12 10/12
Technical Assistance Contact the PolyPlus assistance service via: Internet address: www.polyplus-transfection.com Email: support@polyplus-transfection.com Telephone: + 33 (0) 3 90 40 61 87 Related compounds In vivo-jetpei for in vivo applications References 1. Boussif O., F. Lezoualc'h, M. A. Zanta, M. D. Mergny, D. Scherman, B. Demeneix and J. P. Behr (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci U S A 92, 7297-301 2. Remy J. S., B. Abdallah, M. A. Zanta, O. Boussif, J. P. Behr and B. Demeneix (1998) Gene-Transfer with Lipospermines and Polyethylenimines. Adv. Drug Delivery Rev. 30, 85-95 3. Horbinski C., M. K. Stachowiak, D. Higgins and S. G. Finnegan (2001) Polyethylenimine mediated transfection of cultured postmitotic neurons from rat sympathic ganglia and adult human retina. BMC Neuroscience 2, 1471-2202 4. Lambert R. C., Y. Maulet, J. L. Dupont, S. Mykita, P. Craig, S. Volsen and A. Feltz (1996) Polyethylenimine-Mediated DNA Transfection of Peripheral and Central Neurons in Primary Culture - Probing Ca2+ Channel Structure and Function with Antisense Oligonucleotides. Mol. Cell. Neurosci. 7, 239-246 5. Mislick K. A. and J. D. Baldeschwieler (1996) Evidence for the role of proteoglycans in cation-mediated gene transfer. Proc Natl Acad Sci USA 93, 12349-12354 6. Behr J. (1996) L'éponge à protons: un moyen d'entrer dans une cellule auquel les virus n'ont pas pensé. Medecine&Science 12, 56-58 7. Behr J. P. (1997) The Proton Sponge - A Trick to Enter Cells the Viruses Did Not Exploit. CHIMIA 51, 34-36 8. Boussif O., M. A. Zanta and J. P. Behr (1996) Optimized Galenics Improve in-vitro Gene-Transfer with Cationic Molecules Up to 1000-Fold. Gene Therapy 3, 1074-1080 11/12 12/12