VDL102.3 Production of Adenovirus in 293 Cells

Transcription

1 1. Purpose CENTER FOR CELL & GENE THERAPY 1.1. The purpose of this protocol is to produce adenoviral vectors from transfected plasmid DNA This procedure is routinely performed in the Vector Development Laboratory (VDL) following Good Laboratory Practices (GLP). 2. Abbreviations and Definitions 2.1. SOP Standard Operating Procedure 2.2. VDL Vector Development Laboratory 2.3. GLP Good Laboratory Practice 2.4. BSC Biological Safety Cabinet 2.5. Antibiotics Penicillin/Streptomycin/Anti-mycotic Solution 2.6. DMEM Delbecco s Modified Essential Medium with 1% Antibiotics 2.7. FBS Fetal Bovine Serum 2.8. DMEM10 DMEM with 10% FBS, 1% Antibiotics 2.9. DMEM2 DMEM with 2% FBS, 1% Antibiotics MEM 2X Modified Eagle s Medium MEM20 MEM with 20% FBS, 2% Antibiotics PBS Phosphate Buffered Saline without Ca 2+ and Mg BSA Bovine Serum Albumin HBS HEPES Buffered Saline TE Tris-Ethylenediaminetetraacetic Acid 3. Equipment, Materials, and Reagents NOTE: All materials in contact with cells must be sterile, pyrogen-free and used according to the manufacturer s directions unless stated otherwise. Equivalent materials and equipment may be used but all changes must be recorded in the laboratory notebook Equipment BSC ºC/5% CO 2 incubator Pipette aid Pressure Cooker Water bath set at 45ºC Water bath set at 37 ºC Inverted microscope with 20X objective Page 1 of 7

2 Microcentrifuge Vortex 3.2. Materials mm dishes Corning Serological pipets Corning Sterile pipet tips VWR ml tubes Axygen Cotton-plugged Pasteur pipets VWR Latex bulb for Pasteur pipets VWR Glass bottles Corning 3.3. Reagents DMEM Hyclone DMEM10 VDL DMEM2 VDL MEM Invitrogen MEM20 VDL PBS Invitrogen FBS Atlas Antibiotics Cellgro SeqPlaque Agarose Cambrex % Sterile Agarose VDL Deionized Water Baxter PacI New England Biolabs X Restriction enzyme buffer 1 New England Biolabs X BSA New England Biolabs Phenol/Chloroform/Isoamyl Alcohol Invitrogen NaOAc Sigma M NaOAc VDL Absolute Ethanol AAPER % Ethanol VDL % Ethanol VDL mg/ml Glycogen Roche Profection Mammalian Transfection Kit Promega X TE buffer, ph 8.0 VDL Page 2 of 7

3 3.4. Starting Materials CENTER FOR CELL & GENE THERAPY , 60 mm dishes of 40-50% confluent 293 cells Plasmid DNA, 10μg 3.5. Test Sample Identification The bar code on the plasmid will be scanned and compared to the computer database to ensure the correct sample is being processed One copy of the barcode will be printed and applied to a copy of this SOP for the final record. 4. Procedure Day 1 Preparation of Plasmid 4.1. In a 1.7 ml tube, combine 10 μg of plasmid DNA, 1 μl of PacI, 3 μl 10X restriction enzyme buffer 1, and 3 μl 10X BSA. Q.S. the reaction to 30 μl with sterile water Spin reaction briefly in a microcentrifuge to collect reaction components at the bottom of the tube Transfer tube to a 37ºC water bath for overnight incubation. Day 2 Preparation of Cells 4.4. Check the cells to ensure they are at the proper density. If not contact laboratory director Three hours prior to transfection, aspirate the media from the cells and replace with 5 ml DMEM Return cells to 37ºC/5% CO 2 incubator until use. Preparation of Kit Reagents 4.7. Remove Profection Mammalian Transfection Kit from the -20ºC freezer and allow reagents (sterile water, CaCl 2, 2X HBS) to warm to room temperature. Preparation of Plasmid 4.8. Add 70 μl sterile water to the overnight digest to bring the volume up to 100 μl Add 100 μl phenol:chloroform:isoamyl alcohol to the PacI digested DNA Vortex thoroughly Spin the tube in a microcentrifuge at 14,000 rpm for 5 minutes to separate the phases Carefully transfer the top aqueous layer to a clean 1.7 ml tube. Discard the interface and lower phase into an organic waste container. Page 3 of 7

4 4.13. Add 400 μl ice-cold 95% EtOH, 50 μl 3 M NaOAc, and 1 μl glycogen to the retained aqueous layer Vortex thoroughly Spin the tube in a microcentrifuge at 14,000 rpm for 5 minutes A white DNA pellet should be visible on the bottom of the tube Carefully remove and discard the supernatant Wash the pellet with ice-cold 70% ethanol Vortex thoroughly Spin in a microcentrifuge at 14,000 rpm for 5 minutes The DNA pellet should again be visible on the bottom of the tube Carefully aspirate off the supernatant Air dry the pellet for approximately 15 minutes at room temperature to evaporate residual ethanol When the pellet is dry, dissolve the DNA pellet in 10 μl sterile 1X TE buffer and store at -20 C until use. Transfection NOTE: Perform all steps in a certified BSC, using aseptic technique Mix kit reagents thoroughly by swirling the container or vortexing For each transfection, prepare the DNA and 2X HBS solutions in separate tubes The transfections will be performed with 2 concentrations of DNA, 4 μg and 6 μg Set up the reactions as follows: Transfection Reaction 4 μg DNA 6 μg DNA Tube 1 (μl) (μl) Linear plasmid M CaCl Sterile water Tube 2 2X HBS Total Page 4 of 7

5 4.29. Gently vortex the tube containing the 2X HBS. Note: The speed should be adjusted such that the tube can be vortexed safely with the cap off and can accommodate the addition of the prepared DNA solution Continue to vortex while adding the prepared DNA solution in tube 1 dropwise Incubate the solution at room temperature for 30 minutes Vortex the transfection solution again just prior to adding it to the cells Add the solution dropwise to the cells Gently swirl the dishes to distribute the precipitate evenly over the cells Return the plates to the 37ºC/5% CO 2 incubator for hours. Day Melt 2% agarose in the pressure cooker and transfer to 45ºC water bath Place MEM20 in 45ºC water bath Once the agarose and MEM20 reach 45ºC, transfer the bottles to the BSC Prepare a 1:1 solution of MEM20:agarose. Each 60 mm dish will require 4 ml of overlay Aspirate media from cells and wash cells gently with PBS Overlay the cells with 4 ml overlay media and allow overlay to solidify at room temperature for 30 minutes Transfer cells to 37ºC/5% CO 2 incubator. Days Every 3-4 days, prepare overlay as described in steps For the subsequent overlays, only 2 ml of the overlay solution is required per 60 mm dish Examine the cell monolayers under the microscope daily for the formation of plaques. Note: Plaques will begin to form around 7 days post-transfection. It is best to pick plaques early than wait since they will continue to grow and overlap. Plaques can be picked over a several day period Select plaques to pick by looking at the monolayer under the microscope When a plaque is found, mark the site of the plaque on the bottom of the dish with a pen Transfer the dish to the BSC Label one 1.7 ml tube for each plaque selected. Label the tubes with the date (month and day) and the plaque number. For example the first plaque selected on February 10 will be labeled Add 0.5 ml DMEM10 to each tube Using a cotton plugged Pasteur pipet with latex bulb, aspirate the plaque by inserting the pipet at the site of the marking on the bottom of the dish. Prior to inserting the pipet into Page 5 of 7

6 the monolayer squeeze the latex bulb. Insert the pipet passing through the overlay to the monolayer. When the tip of the pipet reaches the dish, release the latex bulb and the agar plug will be sucked into the pipet Transfer the tip of the pipet to the respective tube and pipet the media in and out of the pipet. The media will wash the plaque out of the pipet and into the tube Repeat plaque selection and picking until 6-8 plaques have been collected Store plaques in the -80ºC freeze until ready to expand. 5. Data Collection and Management All data will be collected in a laboratory notebook Deviations of the protocol will be recorded in the laboratory notebook and on the Lab Meeting Sheet. 6. Review and Revisions Written by: Director, VDL Director, Vector Production Director, QA/QC Date Issued: 12/11/2006 Replaces VDL Annual Review: 2011 Reviewed without changes Changed and this version archived QA/QC by: Date Issued: 7/01/2011 Replaces VDL Page 6 of 7

7 Reviewed without changes Changed and this version archived QA/QC by: Date: 2013 Reviewed without changes Changed and this version archived QA/QC by: Date: Page 7 of 7