High-throughput method for screening polymer transfection reagents
The development of a safe and effective gene delivery system is a prerequisite for the success of clinical gene therapy. Despite the high transfer efficiency of virus carriers, researchers have begun to look for non-toxic carrier materials to replace viruses due to safety and virus immunogenicity considerations (Nishikawa and Huaga 2001; Check 2003). Polycations have great potential as one of these non-toxic carriers (De Smedt et al. 2000; Merdan et al. 2002; Wagenberg 2004), which can form nanoscale complexes with plasmid DNA by electrostatic interaction, and in some cases these complexes are effectively absorbed by cells and have good transfection efficiencies. Author: T. Friedman et al, Translator: Jingwei et al, This experiment is from "Gene Transfer".
Operation method
High-throughput method for screening polymer transfection reagents Move High-throughput method for screening polymer transfection reagents Materials reagents C0 S7 cells, a renal fibroblast cell line from African green monkeys, are suitable for transfection with vectors that require expression of the SV40 T antigen (ATCC: CRL-1651) Dimethyl sulfoxide (DMS0, >99. 7% sterile filtration) (Sigma-Aldrich) DMEM medium [50 ml of fetal bovine serum (FBS) and 5 ml of Penicillin-Streptomycin per 500 ml (Invitrogen 21063-029, 10082-147, 25200-056) Honey Bug Stem Cellulase (Promega) p C M V - Luc D N A (l mg/ml aqueous solution, stored at 20°C) (ElimBiopharmaceuticals) Branched PEI, 25 kDa molecular mass (Sigma-Aldrich) Sodium acetate solution (3mol/L, p H 5.2, 0.2um membrane filtration) (Sigma-Aldrich) Membrane protease (Invitrogen 25200-056) Instrumentation 12-channel pipette (V&~undefinedP Scientific), autoclavable Centrifuge tubes (15 ml, sterile) 96-well plate (Corning CJstar 3695), sterilized by UV irradiation for I h on a cell culture ultra-clean bench with laminar flow. Cell counting plates (VWR) Incubator (37°C, 5% C 〇 2; FormaScientific) Small centrifuge tubes (1.5 ml, sterile; Eppendorf, VWR) Micropipettes (Eppendorf) 5~50ul 1 2 channels 50~300ul 1 2 channels 100~IOOOul single channel 2 0 ~ 200ul 1 single channel Vortex oscillator (optional) Pipette tips, autoclaved for 30 min 1 to 200ul 4 cartridges (U.S. Scientific) 100 ~ IOOOul (U.S. Scientifio) Pipette, sterile (VWR) Cell plates (96 wells) Multiwell flat-bottomed plates with lid (2; BDFalcon, sterile, BD 353072) Multi-well plate, 96 wells (2.4mL), V-bottom, Greiner Polypropylene (Sigma-AldrichM1561) Tissue Culture Plate, white 96-well plate (sterile; Corning Costar 3917) Biotinidase plate counter (Perkin Elmer) Timer (VWR) Tissue Culture Filter, 500 ml, 0.2 um cellulose acetate membrane (sterile; Nalgene) Water bath, room temperature Methods All operations must be done in a laminar flow cell culture ultra-clean bench, and all reagents and equipment are sterilized. It is assumed that the readers are familiar with basic sterile cell culture techniques, including cell growth, passaging and inoculation of cells. 1. Inoculate the 96-well white cell culture plate with CIS-7 cells at 1.5 X IO4 per well one day prior to transfection. 2 . Prepare a 100 mg/m l polymer solution by dissolving 20 mg of different polymers in 200 ul of DMSO in a sterile small centrifuge tube. A 100 mg/m l solution of polymer was prepared by dissolving 20 mg of different polymers with 200ul DMSO in a sterile small centrifuge tube. lOmg/m l solution of polymer was prepared by dissolving 100 mg/m l of polymer with Iml DM SO as a positive control. 3 . Dilute 4.2 ml of sodium acetate solution with 495.8 ml of distilled water, prepare 500 ml of 25 mmol/L sodium acetate buffer (pH 5.2), and remove bacteria by vacuum filtration through a Nalgene tissue culture filter. 4 . Pipette 30 ml of 25 mmol/L Sodium Acetate Buffer into a pipetting basin and add 76ul and 80ul of the buffer to rows A and B of the BD Falcon 9 6-well plate, respectively, using a multichannel pipette gun. Following this procedure, the final polymer to DNA ratios will be 20:1 and 100:1, respectively, and 900ul of buffer per well will be added to row A of the Greiner 96-well V-bottom plate. 5 . Thaw the pCMV-Luc DNA reservoir in a room temperature water bath and dilute 600ul lm g/m l of DNA reservoir with 9.4 ml of 25 mmol/L sodium acetate buffer in a 15 m l sterile centrifuge tube. Transfer the diluted D N A solution to a pipetting basin and add 25ul per well to a 96-well plate ). 6- Warm the culture solution in a 37°C water bath for 15 min. transfer the culture solution to a pipette and add 200u l of culture solution per well to a BD Falcon 9 6-well plate. 7- In row A of the Greiner 9 6-well plate (containing 900ul of sodium acetate buffer), add lOOmg/m l of polymer/DMSO solution and the positive control accordingly, 100ul per well (Figure 1, plate 1). Mix with a pipette gun to ensure uniform dissolution of the polymer. All of the following transfers were performed with a 12-channel pipette gun. 8 . Prepare Polymer Diluent (Figure 1, Step 1). a. Transfer 24ul of Polymer Solution from Row A of the Greiner 9 6-well plate to Row A of the Polymer Solution Dilution Plate containing 176ul of Sodium Acetate Buffer. b- Transfer 120^1 of Polymer Solution from Row A of a Greiner 96-well plate to Row B of a Polymer Solution Dilution plate containing 80ul of Sodium Acetate Buffer. c. Blow on the solution with a pipette to ensure that the solution is well mixed. 9 . Prepare the Polymer-DNA Compound (Figure 1, Step 2) a. Transfer 25 m1 of Polymer Solution from row A of the Polymer Solution Diluent Plate and add it sequentially to the ABCD rows of the DNA plate containing 25 ul of DNA. b. From row B of the Polymer Solution Diluent Plate, transfer 25 W of Polymer Solution and add sequentially to rows E, F, G, and H of the DNA plate containing 25 4DNA. c. Time the transfer for 5 min. Repeat each transfer with the pipette gun to ensure uniform mixing of the solutions and to promote polymer-DNA binding. Change the tip of the gun before each addition to prevent contamination between wells. Mixing technique is important to obtain reproducible transfection results. After 10.5 m in, aspirate 30 M1 of polymer-DNA complex from row A of the DNA plate and add it to row A of the plate containing 200 ul of culture medium per well (Figure 1, step 3), and repeat with the other rows sequentially. For each addition, blow several times with the pipette gun to ensure that the solution is well mixed. Replace the tip of the pipette gun each time you add a sample to prevent contamination between wells. 11 - Add the polymer-DNA complex to the cells (Figure i, step 4). a. Aspirate the entire cell culture plate with a 12-channel pipette to ensure that the culture solution is aspirated. b. From Row A of the plate, aspirate I50 mI of polymer-DNA complex into Row A of the cell culture plate. c- Repeat sequentially for the other rows. Replace the tip of the gun for each addition. When adding samples, the tip of the gun is added at an angle to the cell culture plate, along the wall of the well, avoiding adding samples directly above the cells to reduce the liquid flow and vortex force caused by the addition of samples, thus avoiding the suspension of the cells. d. Place the cells back into the incubator and time the incubation for lh. 12. I h later, aspirate the polymer-DNA solution from the cells, add 105ul of fresh culture medium to each well, and continue to incubate the cells. 13-Analysis of fluorescein protein expression. The analysis of fluorescein protein expression is usually carried out 1~5d after transfection, for CO&7 cells, 3d after transfection is more appropriate. a. Thaw the Bright Glo Luciferase Assay Kit in a room temperature water bath for at least lh. b. Add the buffer to the substrate tube, cover, invert several times to mix, and transfer to the pipetting basin. c- Using a multichannel pipette gun, add 100ul of solution to each well of the cell plate, counting 2 m in , shake the plate gently, or mix with a vortex shaker. d. After 2 min, measure the fluorescence intensity on the plate reader at time Is. Correspond to the standard curve and calculate the amount of fluorescein protein expression. For more product details, please visit Aladdin Scientific website.
Bright-Glo Kits (Promega)