This chapter describes in detail the preparation, amplification and large-scale production of helper virus-dependent adenoviral vectors (H D A d ) for use in gene transfection and gene therapy. In this process, 116 production cells suitable for suspension culture, A d N G 163 adjuvant viruses and adjuvant virus-dependent viruses in the p A 28E 4 backbone were used. To date, the improved methods and reagents described in this paper are the most effective for high-throughput, high-quality production of H D A d . The paper also describes the use of standard molecular biology techniques to characterize the physical titer of H D A d , the contamination of the helper virus, and the structure of the genome. Author: T. Friedman et al, Translator: Wei Qin et al. This experiment is from "Gene Transfer".
Operation method
Rescue, amplification, and mass production of helper virus-dependent vectors Move Rescue, amplification, and mass production of helper virus-dependent vectors Materials CaCl2 (2.5m o l /L ) Filter with 0.2um membrane to remove bacteria, dispense in sealed conical tubes and store at 4°C. Cesium chloride gradient solution (1.25 g/m l and 1.35 g/m l ) For I. 25 g/m l and I. 35 g/m l solutions, 54.Og and 70.4 g of solid CsC l were added to 146.Oml and 129.6 m l of dialysis buffer, respectively, and filtered through a 0.2/xm filter. 1.00 ml of this buffer was used for concentration testing. 2 X lemon salt 270m m o l / L K C l 30m m o l / L sodium citrate Sterilize for 45 min at 121°C in an autoclave. Analyzing buffer (l0 m m o l /L Tris-HCl, p H 8.0) Sterilize for 45 min at 121°C in an autoclave. DNAase I Prepare IO ml of a solution containing 20 mmol/L T ris-H C l and IOOmg bovine pancreatic DNA enzyme (PH 7.4), 50 m m ol/L NaCl, lirnn o l/L m T < ! >, O.lm g/m l bovine serum albumin, 50% (VVV) glycerol, stored at -20 °C. Ethanol (70% and 95%, V/V) Ethacridium bromide Fetal Bovine Serum (F B S ) , heat inactivation L-Glutamine Glycerol Autoclave at 121°C for 45 min to sterilize. HDA28E4LacZ This H D A d vector contains an mCMV-fccZ expression cassette H E P E S buffered saline (H B S ) 21m m o l / L H E P E S 137 m m o l / L N a C l 5 m m o l / L K C l 0-7 m m o l / L N a 2 H P O 4 5.5m m o l /L glucose Adjust p H to 7.I with NaH and filter through a 0.2-um filter to remove bacteria. The filtrate is stored at 4°C in tightly closed plastic tubes. The pH of the HBS solution is important for transfection efficiency. Chloramphenicol Magnesium chloride (2m o l /L ) Pressure sterilization at 121°C for 45 min. Minimum Essential Medium (MEM; Invitrogen 61100) P B S + + (Phosphate Buffer Salt Water) Add 0.O l v/v of sterilized MgCl2 and 0.01 v/v of CaCl2 at 68 m m ol/L to PBS. Penicillin/Streptomycin p H D A d Streptavidin solution A 20m g/m l streptavidin solution was prepared with 1 0 mm0 1/L Tris-HCl (pH 7. 5) solution. The solution was held at 56°C. Streptavidin-SDS solution A solution containing 0.5% SDS, 10 mmol/L Tris-HCl (pH 7.4) and 10 mmol/L EDTA (pH 8.0) was used to make a 0.5 m g/m l solution of streptavidin. R N a se A C 10m g/m l) Salmon Sperm DNA (2ug/ul in T E buffer) Sodium deoxycholate (5% W /V ) Bacteria were removed by filtration through a 0.2uL filter. Sucrose solution (40 % W/V) Bacteria removal by filtration through a 0_ 2^m filter TE lm m o l/L E D T A (p H 8. 0) Pressure sterilization at 121°C for 45m in to remove bacteria. T ris -H C l (100 m m o l/L, p H 8. 0) Virus Lysis Buffer Add SDS to the TE buffer to a final concentration of 0.1 % (W/V). 1 % (W/V). Instrumentation Beckman S W 40 T i turntable and ultra-clean centrifuge tube (342413), Beckman S W 35 turn head and ultra-clean tubes (344057) Petri dishes (60 m m and 150 m m ) Electromagnetic stirring (Bellco Glass 7785-D 200) Slide--A-Lyzer Analyzer Card (Pierce 66381) Southern Blot Hybridization Materials and Reagents Spectrometers and cuvettes Rotary flasks (Bellco Glass Company): 250m l (1965-61002) and 3L (1965-61030) METHODOLOGY Monolayer culture 1 1 6 Cell lines 116 Cells were cultured in 150 mm dishes with MEN and 10% heat-inactivated FBS, 0-l m g / m l thiomycin, l O O U /m l penicillin/streptomycin, and 2 m m o l /L L-glutamine. When the cells reach 90% confluence, divide the cells into two (every 2 days) or three (every 3 days). Remember to warm the medium to 37°C before use. 1. Remove the medium. 2. Tap the side of the Petri dish to isolate the cells. 3. Take an appropriate volume of fresh medium, suspend the cells in it, and control the temperature at 37°C and place it in the new medium. In general, transfect 116 cells 2d after cell isolation to ensure adequate cell exposure. A single 116 cell spread in a 15-mm dish can be cultured in 20 60-mm dishes. Preparation of HDAd The p H D A d was first constructed . We used traditional molecular biology methods to insert the expression cassette of interest into P A 28E 4 . In order to package efficiently, the genome of H D A d needs to be 37.8 ~ 27.7k b (B ettetal.1993; Parks et al.1993; Parks et al.1993). 8 ~ 27. 7k b (B ettetal.1993; Parks and G r a h a m 1 9 9 7 ) . The rest of the DNA genome is not intended to contain the restriction endonuclease site used to cleave DNA from the plasmid. For additional information on the design of HDA, please refer to the Preface section. High-quality DNA plasmids are critical for efficient vector preparation, so we used CsCl ultracentrifugation to purify the plasmids (NgetaL 2002b ). The following steps deal with the preparation and amplification of H D A d . For scaling up, the appropriate vectors need to be added. 4- Dispense 116 cells into 60 m m Petri dishes (one vector per dish) and transfect when 70 % confluence is reached in 2d. One hour before transfection, remove the medium and add 5 ml of fresh MEM and 10 % FBS. 5- Digest lOugpHDAd in a volume of 25 to 50 M1 using the appropriate restriction endonuclease. 6. Incubate at 65°C for 20m i n . a- Use digested DNA immediately or store at 4°C and use the next day. b. Examine 0. 5M 1 digested D N A by agarose gel electrophoresis while staining with diaminodiamine ethylphenidate bromide. Complete digestion should produce only two bands: the H D A d genome and the viral plasmid sequence. 7. Add 5 nets of salmon egg DNA to 0.5 ml of HBS buffer through a polystyrene tube. Stir l m i n at maximum speed. 8 . Add the digested pHDAd and carefully mix completely. 9- Add 25fJ 2. 5m o l / L of Ca C l 2 drop by drop, mixing as you go. 10. Incubate for 30 min at room temperature. 1 1- Add 0-5 m l of p H D A d solution dropwise to the 1 1 6 monolayer, but do not remove the medium. 12-Shake the petri dish to distribute the precipitate evenly and incubate overnight. 13- The next day, remove the culture medium. 14. Wash twice with Im l of MEM containing 10 % FBS. 15. Immediately transfect the cells with AdNG163, adding 5 pfu/cell and PBS to bring the volume to 0.lm l. 16- Incubate for lh, shaking the dish every IOmin. 17. Add 2. 5 ml MEM and 5 % FBS. 18. 48 h later, test the cells for all pathological effects (CPE). 9 0 % of the cells should become round and detach from the dish. If this is not the case, look at the end of this chapter for the solution to the problem. I9 - Scrape the cells into the medium and place them inside a small bottle. Add 0.1 v/v of 4 0 % dextrose solution and store at 1 80°C . Remove 0.5 ml of the cell suspension for DNA extraction and test for vector amplification (see step 26). HDAd amplification 20-Thaw cell suspension at 37°C to equilibrate at 37°C. Mix lysis. 21. Co-transfect 90 % of 60 m m dish 116 cell line solution with helper virus and HDAd at multiple transfection concentrations of 2P fu/cell and 0 -4 m l of transfected cell suspension. 22. React for I h in the incubator, shaking every IO m i n . 23. I h after transfection, add 2.5 ml of MEM containing 5 % FBS to the monolayer. 24. Repeat steps 18 to 23 until the HDAd titer is maximized. The number of cycles may vary. HDAd Amplification Assay . We generally amplify in parallel HDAd that can accommodate expression cassettes (e.g., H D A 2 8 E 4 L a c Z ; Fig. 3 B ) because the titer of this vector can be very easily and rapidly detected by X -g a l staining in the 2 9 3 cell line (N g et 25. The titer of contained carriers was determined by X -g al staining in the titer of the 2 9 3 cell line (N g et al, 2002a). 26. the H D A d vector amplification of interest is obtained. a. Centrifuge the cell suspension (obtained in step 19) in a small centrifuge, centrifuge at 750 g for l m i n , and remove the supernatant. b - Resuspend the cell pellet in streptavidin-S D S solution and incubate overnight at 37°C. c - Add 0 .5 m l of 9 5 % ethanol. Let the tube be inverted and mix the solution until a precipitate is seen to form. d. Centrifuge l m i n a small centrifuge at maximum speed to allow D N A to form pellets. e. Wash the DNA twice with ethanol and dry. f. Resuspend the DNA at an appropriate volume of TEF. g - Add DNA at 65°C and stir until dissolved. h . Digest sample volume and digest with appropriate restriction enzyme. i- Detection by agarose gel electrophoresis and staining with brominated diammine ethylphenidate. If the HV- and HDAd-specific bands can be clearly distinguished, it means that the titer of HDAd is maximized (Fig. 3D). Note that the HV-specific band is what we would expect to see, not contamination, because the HV genome does not contain a packaging signal; this can be detected by Southern blotting (Fig. 3D). If this phenomenon is seen in several steps, take the first result that shows this phenomenon, as the probability of vector or helper virus rearrangement increases with each step. If this result is not obtained, repeat the procedure. 27. 9 0 % of a 60 m m dish 116 cell line solution was co-transfected with the helper virus and H D A d in 0. 5 m l of bacteriophage solution at a multiple transfection concentration of 2 pfu/cell and containing the maximum H D A d titer. . 2 8 . After 4 8 h of transfection , the cells were examined for complete various cytopathic effects, and the cells were scraped into the culture medium. 29.750 g Centrifuge the cell suspension for 5 m i n . Remove the supernatant. 30. Resuspend the cell pellet in Im l 100 m m o l / L Tris-HCl (p H 8 , 0 ) solution and 10 % glycerol and freeze at 80°C. Large-scale production of HDAd Co-transfected 116 cells with helper virus and high titer H D A d, co-grown in suspension. 31. The 116 cell line was prepared as described below: a. Transfer the spent medium containing 116 cells from 8 confluent Petri dishes to a 3L fine bottle. b. Tap the petri dish to separate the 116 cells. Divide the cells and remaining culture medium into 3L fine bottles. c. At 37°C, add M E M and 5 % heat-inactivated F B S, 0 -l m g /m l of chlortetracycline, l O O U /m l of penicillin/streptomycin, and 2 m m o l /L of L -glutamic acid to make a final volume of 1L. Tighten all lids. d- Incubate overnight at 37°C on a 60r/m i n magnetic blender. Cell growth in fine bottles does not require a humid, CO2-filled environment. e. Add 0.5L the next day and continue to incubate overnight. f. Add 0.5L the next day and continue overnight incubation. g. Add 1L the next day and continue to incubate overnight. The final volume is 3L. Cell density increases daily, which can be determined by counting before adding medium. 32-Count the number of 116 cells in the suspension as described below: a. Add 2m l of cells to a 15m l conical tube. b. Add 2m l 2 X catalase at 37°C. Mix for 10 s at maximum setting. c - Incubate at 37°C for 5 to 100 m i n . d. Mix for 10 s at maximum setting. e- Measure using a hemocytometer to get two independent cell counts. If these two cell counts are very different, re-measure. And make sure that there are no large clusters of cells that cannot be counted. The cell density should be 2 X 105 ~ 4 X 105 cells/ml. f- Take 0 -I m l of cell suspension in 3L of medium into one well of a 24-well dish and add I m l of fresh medium. These samples serve as controls for the healthy state of the cells without transfection. These samples will, after a few hours, back-patch and re-form a monolayer of cells. 33. large-scale co-transfection of 116 cell lines: b- Resuspend the cell pellet with 100 ml of spent medium and transfer to a 250 ml fine bottle. c. Co-transfect the cells with helper virus at a transfection volume of 2 pfu/ml and HDA solubilization (step 30). Incubate at 60r/min for 2 h at 37°C. d-Transfer co-transfected cells to 3L vials. Add 0.5L of spent solution and I.5L of fresh These cells reattach to the wall, but round off and leave the wall after 48 h. If not, refer to the problem solving section below. If not, refer to the following problem solving instructions f. Incubate at 600 rpm for 2 h at 37°C. g. Collect co-transfected cells by centrifugation at 750 g for 5 m i n at room temperature. h . Resuspend cells in 15m l of l O O m m o l / L Tris-HCl (p H 8. 0 ) solution. Transfer to a 50m l conical tube, and H D A d - up for purification. This method can also be used: resuspend the cells in 15 m l of 100 mmol/L Tris-HCl (p H 8.0) solution. The cells are resuspended in 15 ml of 100 mmol/L Tris-HCl (p H 8.0) solution with 10% glycerol and stored at 80X for later use. HDAd purification 34. Add 2. O m l of 5% sodium deoxycholate solution to the cotransfected cell solution. The mixture immediately thickens to a gel. 35. Incubate at room temperature with agitation for 30m i n . Identification. 50-aliquot vector and store at 80°C. methodology Physical titer 1. The following precautions are taken to evaluate the physical titer of the vector: Physical titer is the concentration of viral particles in the prepared vector, which is measured by the absorption peak at 260 m n after dissolution of the viral particles (Maizel et al., 1968), corrected for the size of the vector genome (Ng et al., 2002a). The ultimate goal of this method is to detect how much viral DNA is in the vector and to express it as vp/m l . a. Add Viral Particle Buffer to the purified vector sample (Step 49) to a volume of 0.5 ml. Also do a blank control where the Virus Particle Buffer is added to the vector fuser [10m m ol/L Tris-HCl (P H 8 .0) and 10% glycerol] to a volume of 0.5 ml. b. Briefly mix the two samples, being careful not to bubble. c- Incubate the samples at 56°C for 10 mol/L. d-Remixed samples lmol/L. e. Use a blank control as a reference data at 260n m and 280n m. Then detect the absorption peaks at 260n m and 280n m for the carrier sample. The ratio of 260/280, which is the relative purity, should be around 1.3. f. Calculate the absorption peak vp/ml at 260n m using the following equation. Ancillary viral contamination HDAd is always contaminated with helper viruses and it is critical to accurately measure the level of contamination. 2- Analysis of helper virus contamination by Southern hybridization (Fig. 4B). Real-time quantitative P C R analysis can also be used (Palmer and Ng 2003), but is very unstable with bacterial spot analysis (Palmer and Ng 2005). HDAd genome structure 3 - Detection of vector genome structure. a- To extract DNA, a purified vector sample is digested with the appropriate restriction enzyme. b- Determine the genomic structure of H D A d by agarose gel electrophoresis and staining with diaminodiamine ethylbenzoate bromide. s o u t h e r n hybridization is more sensitive (Fig. 4B , Fig. 4 0 . 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The solution was held at 56°C for 1 5 m in, then at 37°C for I h, and stored at 1 20°C. The solution was then incubated for 1 5 m in, then at 37°C for I h, and stored at 20°C.
1 0 m m o l/L T ris -H C l ( p H 8. 0)
The solution becomes cloudy but no lumps appear.
al.2002a). In each process, the titer of control HDAd rose 10 to 100-fold until the maximum titer was generated and the process was detected as expected (Fig. 3C). Amplification of the mostly unreported vector was detected by extracting total DNA from the cells cotransfected in each step by agarose gel electrophoresis, bromination, and the use of a bromoform.
The amplification of most of the carriers without reported genes was detected by extracting total DNA from the cotransfected cells in each step and staining them with agarose gel electrophoresis and brominated bromoethenorphine (Fig. 3D).
In each process, the titer against HDAd rises 10- to 100-fold until a maximum titer is produced and is detected as expected (Fig. 3 0 .
The cell concentration is critical during cotransfection, as this will determine the amount of helper virus added and the yield of H D A d .
a- Centrifuge at 750 red for 5m i n at room temperature, and collect cells from 3L of medium. Keep 0.5L of spent medium.
M E M and 5% F B S (2L total).
e-Transfer 0 - I ml of cotransfected cells to one well of a 2 4-well plate with I ml of fresh MEM and 5% FBS.
Replace the lysate with 200 vp/cell from a 150_ plate as described in step 33, not in step 33c. The use of purified HD Ad instead of crude lysate greatly simplifies production. Improved viscosity.
vp/ml== (peak absorption at 260nm) (dilution factor) (I .IX lO 12) (36V (carrier size in kb))