jetpei Cationic polymer transfection reagent

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1 jetpei Cationic polymer transfection reagent Cat# GDSP10105 (0.5 ml) Cat# GDSP10110 (1.0 ml) Cat# GDSP10140 (4 x 1.0 ml) developed by PolyPlus-transfection Product 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 for Qbiogene by PolyPlus-transfection. Chemical structure: HO-(CH 2 ) 2-(CH 2 -CH 2 -NH) n-(ch 2 ) 2-OH jetpei provides superior in vitro transfection when compared to various cationic lipids and polymers 2. jetpei has been successfully used to deliver genes to various established cell lines as well as to 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. Continuous proton influx also induces endosome osmotic swelling and rupture, which provides an escape mechanism for DNA particles to the cytoplasm 1,6,7. Figure 1: Mechanism of gene transfer in eukaryotic cells 1

2 Reagents Provided jetpei is provided as a 7.5 mm solution in sterile, apyrogenic water (expressed as concentration of nitrogen residues). 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 shipped at room temperature and should be stored at 4 C upon arrival. jetpei is stable for 1 year at 4 C. Quality control Functional analysis: every batch of jetpei is tested by transfection into HeLa, NIH 3T3 and CHO cells. Typically, transfection with a firefly luciferase gene (under the control of CMV promoter) gives 10 9 RLU (relative light units)/mg of protein, which corresponds to ca. 100 ng of luciferase protein. Transfection Efficiencies jetpei is a very potent transfection reagent that can be used for both established cell lines and primary cells 1,3,4. The list of cell lines successfully transfected with jetpei can be found with the corresponding references at Some of the most commonly used cell lines are shown below (Table 1). Table 1: Examples of cell lines successfully transfected with jetpei Figure 2: NIH3T3 cells transfected with jetpei at N/P = 5, staining after 24 h Cell line A549 B16-F10 BHK-21 BNL CL2 C2C12 CHO COS-7 DHD Pro.b EA.hy 926 HEK293 HeLa HepG2 IGROV-1 IMR32 K562 KB MCF-7 MRC-5 NIH 3T3 RAW SH-SY5Y SK-N-AS SKOV-3 T24 Cell type human lung carcinoma, type II pneumocytes murine melanoma hamster Syrian Kidney fibroblast-like murine normal embryonic heptacytes murine myoblasts hamster ovary monkey kidney rat colon carcinoma murine endothelial human embryonic kidney fibroblast-like human cervix epitheloid carcinoma human hepatocarcinoma human ovary carcinoma human neuroblastoma chronic leukemia human mouth epidermal carcinoma human breast adenocarcinoma human foetal lung epithelium murine embryonic fibroblasts murine monocytes/macrophages human neuroblastoma cells human neuroblastoma cells ovarian adenocarcinoma human bladder carcinoma 2 Figure 3: CHO cells transfected with jetpei at N/P = 5, staining after 24 h

3 Transfection Protocols Definition of N/P ratio Effective cell entry requires cationic particles. The ionic balance of jetpei cations and DNA anions should thus be in favour of the former. 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. Not every nitrogen atom of PEI being a cation, electroneutrality of jetpei /DNA complexes is reached for N/P = 2-3. In practice, the best transfection results are obtained for N/P = jetpei is provided 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 2 and can be calculated using the following formula: µl of jetpei to be used = (µg of DNA x 3) x N/P ratio 7.5 Table 2. Volume of jetpei solution and amount of DNA for various N/P ratios. Amount of DNA Vol (µl) of jetpei Vol (µl) of jetpei Vol (µl) of jetpei Vol (µl) of jetpei N/P = 3 N/P = 5 N/P = 8 N/P = 10 1 µg µg µg µg µg µg Transient Transfection of adherent cells Reagent required but not provided 150mM NaCl (sterile) is required to dilute the jetpei and DNA. Cell seeding To obtain optimal transfection conditions with jetpei, the cells should be 50-60% confluent. Typically, for transfection in 24-well plates, to cells are seeded per well, 24 hours before transfection. Refer to Table 3 for the recommended number of cells to seed for different culture formats. Table 3. Recommended number of cells to seed the day before transfection Culture vessel Number of adherent cells to seed Surface area per well or plate (cm 2 ) Volume of medium per well or plate (ml) 96-well 48-well 24-well 12-well 6-well 35 mm 60 mm

4 Preparation of complexes We recommend to use jetpei at N/P = 5. (See Table 2 for other N/P ratios.) The following protocol is for a 24-well plate transfection. (See Table 4 for other culture formats.) Dilute 1µg of DNA into 50µl of 150mM NaCl. Dilute 2µl of jetpei solution into 50µl of 150mM NaCl. Add the 50 µl jetpei solution to the 50µl DNA solution at once Important : do not mix the solutions in the reverse order Vortex the solution immediately and spin down briefly. Incubate for 15 to 30 minutes at room temperature. Add 100µl jetpei /DNA mixture per well and homogenize the mixture by gently swirling the plate. Note: the volume of the jetpei /DNA mixture represents one tenth of the total volume of the culture medium. Transfection experiments are stopped after 24 to 48 hours incubation at 37 C and reporter gene activity assessed. For triplicate transfections, refer to the easy protocol included in the jetpei transfection reagent package. Table 4. Complex preparation for different cell culture formats Culture vessel DNA (µg) NaCl (µl) jetpei (µl) jetpei in NaCl (µl) Total volume of complex per well 96-well well well well well mm mm Factors affecting transfection efficiency Unlike many 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 medium volumes (half the quantity indicated in Table 3) or/and by centrifugation of the culture plate (5 min at 280g at room temperature) 8. If the cell line is particularly sensitive, the transfection complexes can be removed after a 2-4 hour incubation period. Remove the complex-containing medium by aspiration and replace with fresh serum containing medium. 4

5 Transient transfection of suspension cells Cell seeding To obtain optimal transfection conditions with jetpei, seed the number of cells adapted to the culture vessel format as shown in Table 5. Table 5. Recommended number of cells to seed. Culture vessel Number of cells Volume of medium per well or plate (ml) Amount of DNA (µg) Final volume of transfection mixture (µl / well) 96-well well well well well / 35 mm Preparation of complexes and transfection procedure We recommend to use jetpei at N/P = 5. Refer to Table 2 for other N/P ratios. The following protocol is for transfection in 6-well plates. Dilute 2 4 µg of DNA into 100 µl of 150 mm NaCl. Dilute 4 8 µl of jetpei solution (see Table 2) into 100 µl of 150 mm NaCl. Add the 100 µl jetpei solution to the 100 µl DNA solution at once Important do not mix the solution in the reverse order Vortex the solution immediately and spin down briefly. Incubate for 15 to 30 minutes at room temperature. Add 200 µl jetpei /DNA mixture drop-wise onto the serum containing medium per well, homogenize the mixture by gently swirling the plate. Incubate at 37 C and 5% CO 2 in a humidified atmosphere. The following protocol is for transfection in 24-well plates. Dilute µg of DNA into 50 µl of 150 mm NaCl. Dilute 1 2 µl of jetpei solution (see Table 2) into 50 µl of 150 mm NaCl. Add the 50 µl jetpei solution to the 50 µl DNA solution at once Important: do not mix the solution in the reverse order Vortex-mix the solution immediately and spin down briefly. Incubate for 15 to 30 minutes at room temperature. Add 100 µl jetpei /DNA mixture drop-wise onto the serum containing medium per well and homogenize the mixture by gently swirling the plate. Incubate at 37 C and 5% CO 2 in a humidified atmosphere. 5

6 Transfection experiments are usually stopped after 24 to 48 hours and the level of gene activity is assessed. Cells growing in suspension are collected by centrifugation at 400g and then resuspended in the desired medium or buffer. Fast Protocol This protocol is optimised as a one-day time saving procedure, which avoids 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 the 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 plate, add µg of collagen or fibronectin per well. Table 6.Recommended number of cells relative to the culture vessel format Culture vessel Number of cells Volume of medium per well (µl) Amount of DNA (µg) Final volume of transfection mixture per well (µl) 96-well well well Transfection in 24-well plates Preparation of the complexes We recommend to use jetpei at a N/P = 3-5. (Refer to Table 2 for other N/P ratios.) Dilute 1 µg of DNA into 50 µl of 150 mm NaCl. Dilute µl of jetpei solution into 50 µl of 150 mm NaCl. Add 50 µl jetpei solution to 50 µl DNA solution at once Important : do not mix the solutions in the opposite order Vortex the solution immediately and spin down briefly. Incubate for ca. 15 minutes at room temperature. Preparation of cells and transfection After trypsinization, wash the cells once with fresh serum-containing medium, then seed cells in 500 µl medium (with serum) in a sterile tube. Add the 100 µl jetpei /DNA mixture to each tube and immediately gently vortex the mixture. Transfer the cell/dna complex solution into the wells (pre-coated with collagen or fibronectin). Incubate at 37 C with 5% CO 2. Transfection experiments are usually stopped after 24 to 48 hours and reporter gene activity assessed. 6

7 HTS protocol (transfection in 96-well plates) Preparation of the complexes Dilute 200 ng of DNA into 25 µl of 150 mm NaCl. Vortex gently. 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 at once Important : do not mix the solutions in the opposite order Vortex 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 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% CO 2. Transfection experiments are usually stopped after 24 to 48 hours and reporter gene activity measured. Stable Transfection Perform transfection in 6-well plates or 60 mm plates according to the protocol for transfection in adherent cells. Start selection with appropriate antibiotic h after transfection. 7

8 Troubleshooting Problems Comments and Suggestions Low transfection efficiency Optimize the amount of plasmid DNA used in the transfection assay. Use high-quality plasmid preparation, free of RNA (the OD 260/280 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 wellcharacterized reporter gene (e.g.gfp Cat # AFP2200). Decrease the culture medium volume. Centrifuge the culture plates (if the cells can withstand it), usually 5 min at 280g. Cellular toxicity 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 complex jetpei /DNA with the cells. Verify the expressed protein toxicity. 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 free of endotoxins. 8

9 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, 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, 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, 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, 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, Behr J. (1996) L'éponge à protons: un moyen d'entrer dans une cellule auquel les virus n'ont pas pensé. Medecine&Science 12, Behr J. P. (1997) The Proton Sponge - A Trick to Enter Cells the Viruses Did Not Exploit. CHIMIA 51, 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, in vivo jetpei for in vivo applications. Fluorescent and biotin tagged jetpei Related compounds 9

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