Construction of first-generation adenoviral vectors
Construction of first-generation adenoviral vectors
The possibility of immune responses against proteins encoded by viral vector backbones limits the application of transgene vectors for in vivo expression. Nevertheless, A d remains the most efficient and versatile gene delivery system. Here we discuss current methods used to construct, amplify, purify, and characterize first-generation A d vectors.
Author: T. Friedman et al, Translated by W. Jing et al. This experiment is from "Gene Transfer".
Operation method
A d Carrier construction and characterization Move A d Carrier construction and characterization Material reagents Agarose (1 % W ) Dissolve Ig agarose (UltraPure; Invitrogen, 15510-027) in IOOml sterile water. i21°C for 20 min. Store at room temperature. Sterilize for 20 min at 21°C. Store at room temperature and dissolve in microwave before use. Bacteria BJ5 1 8 3 Cells (RecA - Adept) Available in commercial AdEasy kits, see Table 1 for details. D H 5a (R ecA-deficient; In v itro g en , 18258-012) Receptor cells were manufactured using the rubidium chloride method, see Sambrcok et al. (1989) 0 .2 m l fraction stored at 80°C. Saturated phenol solution Cell lines Low-generation 293 cells (Microbix; T o ro n to , C anada, PEMD2-01) A549 cells (ATCC; M anassas, Virginia, CCL-185) 293 and A549 cells were cultured in 150 mm plates at 37°C in a 5% CO2 incubator with 90% confluence and 1:2 or 1:3 passaging. 293N 3S cells (Microbix, PI>02-02) 293N3S cells were cultured in 15 m m plates as described above. Cells were cultured in suspension medium with 70r/m in vibration, and when cells reached 5X105 cells/m l, they were passaged I:2 or I:3. Cesium chloride gradient solution (1.25~1.35 g/m l ) Dissolve 54.O g and 70.4 g of solid cesium chloride (Fisher Scientific, B P 1 5 9 5 -1 ) into 146 ml and 129.6 ml of dialysate, respectively. 0.2 Call! filter. Chloroform: Isopropanol (24 : I , V /V ) I X Sodium citrate 135m m o l / L K C K 15m m o l /L sodium citrate Dissolve in deionized water and sterilize at 121°C for 45 min. 2X sodium citrate 270m m o l / L K C K 30m m o l /L sodium citrate Dissolve in deionized water and sterilize at 121°C for 45 min. Complete medium Minimal essential medium (M E M ; Sigma-Aldrich, M 2279) 10% fetal bovine serum (F B S ; Sigma-Aldrich, F 2442-500m l ) 2m m o l / L G l u t a M A X (Invitrogen, 35050-062) Dialysis buffer (l 0 m m o l / L Tris-H C K ! > p H 8.0) Deionized water 0.2 um membrane filtration. D N A Enzyme I (Sigma-Aldrich, D-5025) Dissolve lOmg/ml of DNAase I in 20m m ol/L Tris-HCl (p H 7 .6 ), 50m m ol/L NaCl, lm m ol/L D T T and 50% glycerol. E B Isopropyl alcohol L B medium M g C l 2 (2mol/L ) Sterilize with deionized water at 121°C for 45 min. Keep the medium M E M (Sigma-Aldrich) 5% F B S (Sigma-Aldrich) 2 m m o l / L G l u t a M A X (Invitrogen) I X Antibiotic-Antifungal (Invitrogen) 2X Maintenance Medium 2 X M E M (Invitrogen, 11935-046) 10% F B S (Sigma-Aldrich) 2X Antibiotic - Antifungal (Invitrogen) P B S 137 m m o l / L NaCl 8.2m m o l / L N a 2 H P 0 4 1.5 m m o l / L K H 2P O 4 2.7 m m o l / L K C l Dissolve in deionized water and sterilize at 121°C for 45 m i n . Plasmids: shuttle and backbone plasmids (Tables I and 2) Restriction endonucleases PacI restriction endonuclease (N e w England BioLabs, R 0547L ) PmeI ( N e w England BioLab, R 05605) Additional enzymes may be required to clone the gene of interest into the shuttle vector. R N a s e A (10m g /m l ; Sigma-Aldrich, R -4875) NaCl (5mol/L ) Dissolve in deionized water and sterilize at 121°C for 45 min. Sodium deoxycholate (5% m/V ) 0 -2um filter. S D S - Protease K Solution 10 m m o l / L Tris-HCl (p H 7. 4) 10 m m o l / L E D T A 1 % S D S (m /V ) l m g / m l Protease K S D S (0.1 % m /V ) - T E Dissolve 0- Ig SDS into IOOml T E . Sucrose (40% m/V ) Dissolve 40 g of sucrose into IOOml PBS solution and filter through a 0.2 um membrane. SuperFect Transfection Reagent (Q I A G E N , 301305) I X T E 10 m m o l / L Tris-HCl (p H 7. 5) l m m o l / L E D T A Sterilize in deionized water at 121°C for 45 min. Sterilize in 0.1 X TE, H2O diluted I X solution at 121°C for 45 min. 1 X trypsin-EDTA PBS dilution 10X solution (Invitrogen, 15400-054). Instrumentation Centrifuge (Avanti J-25 I; Backemann) and JLA 10.500 rotor (Backemann). Heat Sealer (e.g., BECKMANN Model 7700 Cordless Tubing Topper) Hematology (VWR International, 15170-172) Incubator (37 °C, 5 % C02) Ultra-clean table Magnetic Stirrer (#5, Bellco Glass, 7785-D 2005) Centrifuge tube (1.5 ml) Pipette, deionized cotton plug Petri dishes with 1.5% agar gel (granulated gel, Fisher Scientific, BP 1423-500) 25ug/ml kanamycin L B medium. Quick-seal (16m m X 76m m ultracentrifuge tube; B e c k m a n , 344322) Slide--A-Lyzer analyzer cartridge (10 OOOkDa molecular mass, 0.5 to 3m l ; Pierce Rockford, Illinois, 664250) Polypropylene tubing (13m l with cap) Polypropylene tube (5m l ) Stirring bottles with impeller assembly: 250m l (Bellco Glass, 1965-61002) and 3000m l (BellcoGlass, 1965-61030). Syringe (3cc) with 22-gauge needle Ultracentrifuge (e.g., B e c k m a n Optima X L -I O O K Ultracentrifuge) 70.1 Ti rotor (B e c k m a n ) S W 41 T i Floating Bucket Rotor (B e c k m a n ) Ultra-Clear (14m m X 89m m ) Ultra-Speed Centrifuge Tubes (B e c k m a n , 344059) Methods Operation A d must be performed in a laboratory that meets biosafety level 2. Ultra-clean benches and incubators for specialized A d use. Sterilized instruments, reagents, and methods should be used with care. Decontaminate solid and liquid wastes and disinfect contaminated surfaces. Production of infectious A d plasmids 1. Use standard cloning procedures to produce a shuttle vector containing the transgene of interest. 2 . In Total Reaction System 2(^1, digest 2ug of shuttle plasmid with 1ul of Tolu1 and leave overnight at 37 °C. 3. Add 3ul of PMI-digested shuttle plasmid and 3.3ul of 0.lug/ul super-helical pAdEasy to a 13 ml capped polypropylene tube. Add 3ul P m e I shuttle vector without p A d E a s y to another tube as a negative control. Cool on ice. 4 . Melt two 0.2 m l portions of B J 5183 receptor cells on ice. 5 - Add 0. 2m l B J 5183 cells to D N A and incubate on ice for 25m i n . 6.42°C heat shock for 90s, then place on ice for 2m i n . 7 . Incubate each tube with 1ml of LB medium at 225r/min at 37℃ for 25min. 8-Transfer the bacteria to I.5 ml centrifuge tubes and centrifuge at 8500 g for 1 m i n at room temperature. 9 . Remove I m l L B medium and resuspend precipitate with a total of 0. 2m l . 10. Add 0. I m l to 1. 5 % agar gel L B 25M g/m l kanamycin culture plate. One plate was used as a control test. There should be no clones on the negative control plate. However, if the background clones do not appear, there will be significantly fewer recombinant clones than in a shuttle plasmid vector that is not digested. 1 1 . Pick 4 to 8 small clones and purify the plasmid DNA by small volume base lysis. Suspend D N A into 25ul of 0.1 X T E . 12. Repeat steps 5 to 10 with 5ul DNA and IOOlUl DH5a instead of BJ5183. Pick two clones per plate. 13. Purify DNA by small volume alkaline lysis. The DNA is digested with different restriction enzymes to verify the plasmid structure. The correct clones are then centrifuged through a CsCl density gradient and used to generate recombinant A d carrier (step 15). Cell Preparation 1 4 Preparation of cultured cells for transfection Preparation of walled 293 and A549 cells a. Remove the medium from 150 m m plates in which 2 or A 549 cells are cultured. b. Rinse the monolayer twice with 5 ml l X sodium citrate (293 cells) or 2 m l trypsin-EDTA (A549 cells). c- For 293 cells, after the second rinse, remove the rest of the liquid and leave only 0.5 m l of sodium citrate 22°C on the culture plate for 5 ~ lOmin, until the cells are detached from the culture plate. For A549 cells, incubate with 2 ml of trypsin-EDTA at 37°C for 5 min until cells start to detach. d. Tap the wall of the culture plate to dislodge the cells. e- Take cells to 12m l complete medium and dispense into new culture plates. Preparation of 293N3S Cell Suspension a-Transfer 6 full-grown 150m m culture plates of 293N 3S cells into a 3L stirred incubator and add maintenance medium to 1L. b-Transfer 2m l of suspended cells into 15m l polypropylene conical tubes every 1-2d. c. Add 2m l of 2X sodium citrate and shake strongly for l0 s. d. Incubate the cells at 37°C for 15mn. e-Shake vigorously for 10s. f . Serumeter cell count. If the cells are clumped and difficult to count, continue incubation at 37°C with shaking until the cells disperse. g. When the cell concentration exceeds 4X105 cells/ml, add IL to maintain the medium until the cell density is 3X105~5X105. Transfection and Regeneration of A d Vectors 15. Digest the IOug plasmid encoding the recombinant A d genome with 2ul of P acI in a 50ul reaction system at 37°C overnight. 16. Mix 40M1P acI digested DNA with 360M1MEM and 16^1 SuperFect Reagent into a round bottomed 5mL polypropylene tube. Other transfection reagents may be used in addition to SuperFect. 17-Shake vigorously for l0 s. Allow to stand at room temperature for 15 min to form the complex. 18-Add 2. 4 ml of MEM to the complex containing D N A -S u p erF ect. Blow to mix. 19. 2 m l PBS rinse 293 cell-inoculated 35 m m plates twice. 20. Transfer 0 -7m l of plasmid mixture suspension to 3 5 mm 293 cell plates. 21. Incubate the cells at 37°C, 5%C0 2 for 3 h. Incubate the cells for 2 h at 37°C. 22-Approximately 2.5 h later, 1% ONAO agarose solution (at least 1.5 ml per 35 mm plate) was lysed in the microwave oven. Equilibrate in a water bath at 42°C. 23-Equilibrate 2 X maintenance medium (I.5m l per 35m m culture plate ) 37°C . After 24.3 h , remove the transfection solution from the transfected cells and rinse the monolayer with l m l P B S . 25. Mix equal parts of agarose gum solution and 2X maintenance medium. Add 3 m l to each 35 m m plate of 293 cells. This step must be done quickly to avoid curing the agarose gel, but gently to avoid dispersing the monolayer of cells. 26- Allow the overlay to cure (about 15m i n , 22°C ) Return the cells to the incubator. 27-Incubate until spots form (7 to 12d). 28.Select a few well-isolated spots. Use a sterilized pipette to remove the agarose plugs from the spots. Place each stopper into a 4 % sucrose P B S bottle. Shake gently at -80°C. Store. The initial Ad spot isolation requires a secondary spot purification (steps 22 to 28) to ensure that the virus forms a single clone. Analyze the amplified spot-separated A d vector. 29. As obtained above for each purified spot vector, add IOOfiI to two 35 m m long 293 cell plates (90% confluent), and return the cells to the incubator. 3 ○. Allow virus uptake for I h , shaking the plates every 10 to 15 m i n . 31- After the uptake period, add 2m l of maintenance medium per plate and return to the incubator. 32- Examine the Cellular Pathogenic Effect (C PE) of each plate. Cells should be spherical or detached from the plate. Use one plate to analyze reconstituted Ad results. Keep the other plate components for amplification. Analyze spot-separated A d vectors a. -Once complete C P E is obtained, let stand l O m i n an ultra-clean bench to allow shed cells to settle to the bottom. b . Carefully remove the medium. Resuspend the cells with 0. 2m l of S D S ^ proteinase K solution. Transfer the cell suspension to a centrifuge tube. c. Incubate the lysate at 37°C overnight. d-Add 0. 3 m l T E to the lysate. Extract with 0.5 m l saturated phenol solution followed by 0.5 m l chloroform : isopropanol. e-Add 0. I m l 5m o l / L N a C l and 0. 5m l isopropanol. f. Centrifuge at 20,000 g at 4°C for 10 m i n to precipitate D NA. g. Resuspend DNA at 20-50 ul TE and digest with 5-IOfjJ with appropriate restriction endonuclease. h - Electrophoretic detection of the resultant bands on 0.8% agarose followed by E B staining. Amplification of spot-separated A d vectors a. - Once complete C P E is obtained (step 32), scrape cells from the culture plate into complete medium and transfer to a 4 m l freezing tube. b - Add 40 % sucrose P B S to a final concentration of 4 % . Shake gently. Use immediately or store at 80°C for later use. c. Inoculate I5O m m 293 plates with IML and return cells to the incubator. d. Absorb virus for 1 hour. Vibrate plate every 15 m i n . e. After the uptake period, add 20 ml of maintenance medium to each plate and incubate in an incubator. f. Check for CPE. Once the CpE is determined, transfer the cells and medium to 50 ml freezing tubes. Add 1/10th of a volume of 40% sucrose PBS to a final concentration of 4% sucrose and store at 80°C. Bulk Preparation of A d Vectors 33-Melt the inoculum in a 22°C water bath. If desired, increase inoculum volume to 30m l M E M . Preparation of A d Vectors for Adherent Cell 293 Bulk a- Remove medium (approx. 9 0 % fused at infection) from 30 150m m culture plates of 293 cells, 10 at a time, and re-inoculate with I m l inoculum. b. Incubate at 37°C, 5 % C 02 for I h for virus uptake, oscillating every 10 to 15 m i n . c- Add 20 m l of maintenance medium and incubate in a cell culture incubator. d. Detect cells daily until C P E appears (2~3d). e. Transfer cells and medium to two 500m l polypropylene bottles Most of the cells should be dislodged in the medium; cells that are not dislodged can be dislodged by shaking the cell bottle. The cells can be dislodged by shaking the cell flasks. f. Use the same lance to rinse 10 culture plates twice with I O m l of P B S . g. Centrifuge the cells at 650 g for 20 m i n at 4°C. Pour out the medium and retain the cell sediment. h - Resuspend cells with 3m l of 4 % sucrose P B S and transfer to 50m l freezing tube. i. Use the same pipette to rinse the bottle once with 2 n d 4 % sucrose P B S and again with 4 to 5 m l of sucrose P B S. ii. The total amount of cells precipitated should be 15 ml. Cells are either purified directly from the vector or stored at 80°C. Mass Production of A d Vector Using Suspended 293N 3S Cells a- Dispense 3L of stirred cultures of 293N 3S cells into 8 500m l centrifuge flasks. b. Centrifuge at 650 g at room temperature for 20 m i n . Pour medium into I L sterilized bottles. Reserve I L of exhausted medium and add to a 3 l blender. Incubate in an incubator. c. Suspend the cell precipitate using the exhausted medium at a final concentration of 40m l . Transfer the suspension to a 250m l blender. Wash twice with IO m l of exhausted medium and transfer to a stirred incubator. d- Add melted grafted seeds to the incubator. The total amount in the container should be 100 ml. e. Transfer the flask to the incubator and agitate the cells for 2 h at 70r/m i n . f. Transfer the cells to 3L suspension flasks containing IL of exhausted medium. Rinse the 250mL stirred flask twice with 250mL of fresh maintenance medium and transfer to a 3L stirred flask. Add 500 ml of Fresh Maintenance Medium to a final concentration of approximately 2L. g-Transfer 2m l of suspended cells to 35_ culture plate and place in 37°C, 5%(:02 incubator. Cells should be re-adhered to the culture plate. h . Return the suspension culture flask to the incubator. i. When complete C P E appears in the 35m m culture plate (after 2 ~ as), pour out the suspension medium into a 50 ml centrifuge flask. ii. j. Centrifuge at 650 g for 20 m i n at 4°C. Pour off the medium and retain the cell sediment. k . Resuspend the cells with 3m l 4 % cheap sugar P B S and transfer to a 50m i conical tube. l. Use the same pipette to rinse the bottle once with 2m l of 4 % Sucrose P B S and once with 4 to 5m l of 4 % Sucrose P B S . Vector purification 34.37°C Water bath melting of A d carrier precipitates prepared in bulk by both methods. 35. Add 1.5 ml 5% desoxycorticosterone to the precipitate. Incubate at 22°0,300 min with frequent mixing and inversion. The lysate is a thick, highly viscous liquid. 36. Add 0. 3 m l 2m o l / L M g C l 2 , 0.15 ml 10m g / m l R N A enzyme A, and 0.15m l 10m g / m l D N A enzyme I . Incubate at 37°C, mixing occasionally by inversion, for 30 to 60 m i n . Incubate at 37° C. Once the viscous lysate is near water, incubate at 22° C. Incubate at 37° C. and incubate at 30-60 m i n . Incubate at 37°C, mixing occasionally by inversion, for 30 to 60 m i n . 38- Prepare for C s C l gradient centrifugation using Ultra-Clear ultracentrifuge tubes (2 tubes per virus). a. Add 2 ml of I.35 g/ml C s C U per tube. b. Add 1.25 g/m l of C s C U to the upper layer in a small center (at a rate of about 30 s/m l if a steady flow rate is used). c- Carefully add an equal amount of clarified lysate (6. 5 to 7 m l ) to each tube. d- Equilibrate each tube and transfer to a S W 41 rotor centrifuge. 39 - Centrifuge samples at 10°C for l h at 35 OOOr/m i n using a slow acceleration and deceleration program (500r/m i n greater than 5m i n ) . The viral bands were the lowest visible bands in the gradient, at the 1.25 to I.35 g/m l level of the gradient. 40-Use a 3c c needle tube and insert a 22 needle into the tube approximately l c m below the viral band. Gently tilt the needle so that the strip moves slowly parallel to the strip, the strip lowers, and the needle lowers. Viruses from two gradient tubes can be combined into a single Quick-Seal Ultracentrifuge tube. 41- Fill the Quick-Seal tube containing the virus to the neck of the bottle with 1.35 g / m l C s C U Use a heat seal to seal the QuickSeal tube. Prepare equilibration tubes by filling Quick-Seal tubes to the neck of the bottle with I. 35 g/ml CsCl. 42.35 O O Or/min Centrifuge overnight with 70.1 T i Turning head Maximum acceleration Maximum deceleration K T C. Puncture the cap of the sealed Ti tube to create an air population. Use a 3c c and 2 2 gauge needle to puncture the tube under the virus i o n . Bevel the needle so that it parallels the strip, move slowly, lower the strip, and lower the needle. 44.A d Pour into prepared dialysis cassette. Remove air with a needle. 45.Dialyze A d carrier for 2 4 h at 4°C with 500 m l dialysis buffer. 46.Transfer the carrier from the dialysis cassette. Retain syringe and 0.9 m l dialysis buffer. 47- Add 4 0 % sucrose P B S to the carrier and a final concentration of 4 % dialysate. Preserve the purified carrier in small fractions (100 to 2(%1) -80°C and buffer at a 2(T C . Identification of purified A d vectors 48. Further characterization of A d vectors is done by analyzing the genomic structure, testing titers, and detecting the presence of replication-competent complete adenovirus (R C A ) contamination. Determine the genomic structure of purified A d vectors. a. Flush the syringe for transferring the vector from the lysate with ∼0.2 ml of SDS ■ Proteinase K . Transfer the liquid to a 5 ml centrifuge tube. b. Incubate overnight at 37°C. c-Add 0.3 ml of TE to the lysate. Saturate phenol with 0.5 ml of buffer, followed by 0.5 ml of chloroform:isoamyl alcohol extraction. d- Add 0 - I m l 5m o l / L N a C l and isopropanol. e. Centrifuge the starch DNA at 4°C for IOmin at 20,000 μm. f. Resuspend DNA with 20~50^x1 T E and digest 5~lO/xl with appropriate restriction endonuclease. g. 0.8 % agarose electrophoresis for detection of enzyme bands. 0.8 % agarose electrophoresis to detect the enzyme bands, followed by EB staining. Determine the titer of the infectious unit by analyzing the plaque-forming unit (Pfu) using the a. Prepare a serial dilution of A d carrier (10-4 ~I(T s)) in M E M . b- Infect 293 cells in a 6-well plate with 0.1 m l of each fractional dilution (~90 % confluent), and return cells to the incubator. c-Viral uptake for l h with vibration of the plate every 10 to 15 m i n . d-After the uptake period, spread the agar gel as described previously. e-Incubate for 10 to 12d and count the spots. The number of spots per well is multiplied by a dilution factor to determine the vector titer pfu/ml. Determination of carrier titer by particles/m l a. Dilute 20 to 50 M 1 pure carrier with Iml 0 -1 % S D S --T E to a final volume of I m U Use dialysis buffer (in step 4 6) as a blank control. b. Incubate at 56°C for IO m i n , shaking gently and centrifuging gently. c. Determine O D 2m. d. Calculate number of particles/m l , wild-type A d extinction coefficient 1.1 X I O 12 (MaizeletaL 1968). (O D 2w) (dilution factor) (I . I X l O 12) As stated the standard A d carrier should have a particle number to p fu ratio of roughly 10 (Mittereder et al. 1996). To detect the presence of R C A in the prepared purified vector Recombination of vector D N A with A d 5D N A in 293 or 911 cells may result in transfer of E l to the vector (Lochmuller et al. 1904), producing R C A . A 549 does not express E l and thus does not efficiently support replication of E l-deficient vectors. Therefore, transfection with purified A d , once contaminated with R C A , causes C P E in A 549 cells. a. A 60m m plate of A 549 cells (90 % confluent) was infected with 250u l M E M solubilized IO6Pfu. Another 60 m m culture plate was lysed with IO6Pfu 250^x1 M E M . Infect 150 m m plates with I m l M E M lysed with IO8Pfu carrier. The cells were returned to the incubator for culture. b. Virus is absorbed for l h, and the plate is oscillated every 10 to 15 m i n . c. After the uptake period, add 5m l or 20m l of maintenance medium per plate and return to the incubator. d. Once there is significant CPE or after 7d, harvest monolayers by scraping cells into the medium, add 40% prasugar PBS to a final concentration of 4% sucrose and store at 180°C. e- Use I m l medium to lyse harvested virus-infected individual 150 m m A 549± tone-raised plates. Add lm l M E M to the fourth plate as a negative control. f-Viral uptake l h , every 10 to 15 m i n vibration of the culture plate. g. After the uptake period, add 20m l of maintenance medium to each plate and return to the incubator. h . Observe for formation of C P E daily against infected cells and uninfected controls. If necessary, change the medium every 5d. If CPE is evident (which usually occurs about W d after transfection), RCA is present in the purified stock solution. The relative amount of R C A to pfu can be . be determined by comparing the CPE on the 3 infected plates.L DNA was extracted from the plate in which C P E was present. analyzed by restriction enzyme digestion agarose electrophoresis. The left end of the RCA genome will have the same structure as the wild-type A d because of the presence of E l . For more product details, please visit Aladdin Scientific website.
Carnosine (25ug/m l )
Incubate the culture plate so that the cells are 90% confluent before transfection.
The total amount of cell suspension is approximately 15 ml. The cells are either purified directly in the vector or stored at -80°C. The cells are stored at -80°C in the vials.
The total amount of cell precipitation solution presented below is 15m l, scaled up or down depending on the actual amount.
Be careful to extract the smallest amount.
A d Vector storage solution at 180°. < : stable for several years.