Linear polyethyleneimine: synthesis and in vitro and in vivo transfection manipulation experiments
Linear polyethyleneimine (L P E I ) is a very effective transfection reagent suitable for large numbers of cell lines and primary cells, and it is equally suitable for local or systemic gene delivery in vivo. Unlike many other nonviral transfection methods, L P E I has a low dependence on mitosis and can transfect dividing collapsing cells ( B n m n e r et al. 2002). The ease of transfection with LPEI and the fact that the presence of serum in the transfection (not more than 10%) does not significantly reduce the efficiency of the transfectionv but significantly reduces the toxicity, especially for primary cells, is a major advantage of LPEI over most liposomal transfection systems. Optimized use of low doses of L P E I also maintains toxicity at minimal levels. Author: T. Friedman et al, Translated by W. Qin et al. This experiment is from "Gene Transfer".
Operation method
Synthesis of linear polyethyleneimine and transfection experiments Move Synthesis of linear polyethyleneimine and transfection experiments Materials reagents Copper sulfate (analytically pure) Cell culture medium HEPES buffered glucose solution (HBG; 5 % glucose m /V ,20m m o l / L HEPES, pH 7.1) 0.2 um membrane filtration, stored at 4°C. HEPES-buffered saline (H B S ; 150m m o l / L N a C l , 20m m o l / L H E P E S ,p. H 7. 1) Filtered through a 0.2 um membrane and stored at 4°C. Hydrochloric acid (30 %, analytically pure) Mouse, in vivo Phosphorus pentoxide Plasmid D N A Poly(2-ethyl-2-璁唑啉) (5 ○ kDa; Sigma-Aldrich 37, 284-6) Sodium acetate (analytically pure) Sodium hydroxide (50% m/V, analytically pure) Sulfuric acid (30 %, analytically pure) Cells to be transfected Instrumentation beaker (glassware) Brewer's funnel contact thermometer Filter paper 0.2um Filter Ice bath multiwell plate oil bath p H test paper Dispensing funnel Reflux condenser tube Syringe (I m l insulin syringe, 33 gauge needle) U V spectrophotometer 37°C water bath for in vivo experiments Methods Synthesis and purification of L P E I This synthesis requires appropriate safety precautions! L P E I is produced by acid-catalyzed hydrolysis of poly(2-ethyl-2-azoline). Complete depropionylation is critical for the transfection properties of the final product ( T h o m a s et al. 2005). 1- Dissolve 45 g of poly 2-ethyl-2-oxazoline in 200 ml of 30% sulfuric acid. 2. The reaction mixture was heated and stirred for 6d and the resulting propionic acid was removed by azeotropic distillation. 3- The total volume of the mixture was kept constant by adding distilled water dropwise. 4- After 5-6d at 106°C, when no acid vapor is detected by p H paper, the solution is transferred to a beaker and cooled in an ice water bath. Add N2OH while stirring to stop the reaction. Note: The reaction is exothermic! 5. Cool the solution until a precipitate appears. A slower precipitation process is favorable to increase the yield. 6- Filter in a Brewer's funnel with filter paper and wash the precipitate with distilled water until the tap water becomes neutral. 7- Dry L P E I -week with P 2O 5 and store at room temperature. 8. Resuspend IOO mg of L-PEI in I ml of distilled water, adjust p H to 7.1 with a drop of HCl, and dilute to 2 ml with distilled water. 9- Filter the L P E I solution through a 0.2um membrane. After filtration the concentration was checked by copper complex method. 10. Store 50mg/ml of LPEI solution directly or dilute to lmg/ml and store at 4°C. Store at 80°C for extended periods of time. Storage at 80°C extends the useful life. Quantification of LPI U n g a r o et al. (2003) describe the detection of L P E I by the copper complex method. L P E I and the spiked C u II ion form a dark blue copper-ammonia complex. 11. Prepare 200 m l of 0. lm o l / L p H 5. 4 sodium acetate buffer. 12 - Dissolve 23 mg of CuSO4 in IOO ml of the above sodium acetate buffer, stir until clear, and label as Cu(II) solution. 13 - Prepare a standard solution containing 1 to 100^1 m g L PEI in 100^1 distilled water, 5 concentration gradients in two parallel portions, and use distilled water as a blank. 14-Dilute different amounts of the L P E I solution to be tested in a total volume of l ○○ ml. 15- Add 1(%1 C u (II) solution to the above sample, mix and leave at room temperature for 5m i n . 16- Measure the absorbance value of the solution at 285n m using a U V spectrophotometer and calculate the L P E I concentration using a standard curve. Preparation of polyplex 1 7 . Preparation polyplex Calculate the N/P ratio: The average molecular mass of each nucleotide is 330 Da, containing one P, and the average 43 Da in LPEI contains one N. For example, to prepare a polyplex with an N/P ratio of 6 using Iug DNA, LPEI 43X (6/330) = 0.78 mg. For example, to prepare a polyplex with an N/P ratio of 6 using Iug of DNA, LPEI 43X (6/330) = 0.78m g. lm g/m l In vitro transfection a- Dilution of DNAGO1UgAnl) and LPEI (31fxg/ml ), respectively, in equal volumes with H B S, N /P = 6 . b- Transfer L P E I solution to D N A solution and mix up and down rapidly 5 to 10 times with a pipette. c. Allow to stand at room temperature for 20 m i n before use in transfection experiments. Polyplexes prepared with salt-containing buffer solutions will agglomerate as they are left to stand for longer periods of time. In vivo transfection a. Dilute D N A (40(V g /m l ) and L P E I (3K V g /m l ) with H B S in equal volumes, N /P = 6, respectively. Both plasmids and buffer solutions should be free of endotoxin. b- Transfer the L P E I solution to the D N A solution and mix up and down 10 times rapidly with a pipette. c- Leave at room temperature for 20m i n for injection. Transfection 18-Transfection operation Transfection of adherent cells a. 1~2d before transfection, inoculate the cells in 48-well plates. Cell density should be 50 % to 70 %. b- Remove the cell culture medium and replace it with pre-warmed fresh medium (100 ~ 200ul/cm2). c- Add p ○ lyple x (D N A final concentration 0.5~5ug/m l ). Incubate in cell incubator after slight vortexing by hand. d. After 4 h, replace the transfection solution with fresh culture medium. Transfection of suspension cells a. Centrifuge cells in logarithmic growth phase and resuspend them in fresh cell culture medium at a cell density of 3X 105 cells/m l . b. Add 0-5 ml of cell suspension per well of a 48-well plate. Add human polyplex (containing DNAl ~ 5ug), vortex lightly by hand and incubate in a cell culture incubator. c. Add polyplex solution containing 1~5ug DNA, vortex gently, and place in cell incubator. d. After 4 h, centrifuge at 280 g for 5 m i n , carefully remove the supernatant of about 4 0 0 4 , and replenish with I m I fresh medium. Optimal transgene expression is observed at 24 to 48 h post-transfection, but depends on the cell line and the gene being transfected. In vivo transfection b . Aspirate polyplex solution into an Im l syringe (33-gauge needle), expelling the air. Administer the polyplex solution intravenously (200 to 250 ml) within approximately 5 seconds. c. When injecting polyplex into tissue (e.g., tumor), leave the needle in place for at least one minute to prevent leakage. For more product details, please visit Aladdin Scientific website.
LPEI solution corresponds to an N concentration of 23.3 mmol/L.
a-Fixed mice, with tails immersed in a 37°C water bath I m i n dilated veins.