Recombinant herpes simplex virus vector
Recombinant herpes simplex virus vector
The natural physiological characteristics of H S V -I include the establishment of a lifelong latent state in neurons, where the viral gene remains a free molecule. H S V vectors that produce completely non-replicating, non-toxic, long-term expression of the transgene have been constructed, as well as replicable recombinant gene H S V vectors that maintain replicative capacity in specific cells (e.g., cancer cells), and have been utilized in Phase I and Phase II clinical trials in patients with glioblastoma multiforme. Recent studies have focused on the use of H S V vectors for the treatment of non-neurologic diseases. Large-scale systems for producing H S V vectors for latency efficacy and safety testing have also been developed. This chapter describes the construction of recombinant H S V vectors and how they can be used for transfection of various cell types.
are used for transduction of various cell types.
Author: T. Friedman et al, Translated by W. Qin et al. This experiment is from "Gene Transfer".
Operation method
Construction of replicable and replication-defective H SV vectors Move Construction of replicable and replication-defective H SV vectors Materials reagents Cells: Standard Vero African green monkey kidney cells variant 7b (Marconi et al. 1996), Ver0 cells (ATCC: CCL81), or other helper cell lines. These cells are required for the proliferation of HSV-I replicable or replication-deficient viruses. Crystalline violet solution (1 % solubilized in methanol: d H2O, 50 : 50, V A O) is used for the production of HSV-I. DMEM/10% Fetal Bovine Serum (FBS) D M E M medium supplemented with non-essential amino acids 100 U / m l penicillin G lOOug/ml Streptomycin sulfate 2m m o l l / L Glutamine 10% F B S Ethanol (70%) Glycerin H E P E S (l m o l/L , p H 7. 35) Iodixanol (60%; Invitrogen) Prior to use, dilute 60 % iodixanol with PBS to a final concentration of 20 %. Isopropyl alcohol Lipofectamine 2000 (Invitrogen) Lysis buffer (l ○○ mmol/L Tris-HCl pH 8. 0 and lOmmol/L EDTA) Methyl cellulose overlay (I. 0%) In a 500 m l sterilized bottle with a stirring rod attached, 25ug of methylcellulose was added to IOOml of PBS solution pH 7. 5 and the bottle was subjected to high pressure treatment (45 min) in a liquid stream. After the solution was cooled, 350 ml of DMEM containing nonessential amino acids, lOOU/m l of penicillin G, lOO^g/m l of streptomycin sulfate, and 2 mmol/L of glutamine was added. mix thoroughly, and the bottles were placed on a stirrer at 4°C overnight. When the methylcellulose has dissolved, add 50 ml of FBS. PacI restriction endonuclease (New England BioLabs) PBS CpH 7. 5) 135 mmol/L NaCl 2.5 mmol/L K C l I.5 mmol/L KH2P0 4 8.Ommol/L Na2 HP 0 4 Plasmid Transfection plasmid p l i u i (Krisky et al. 1997) Plasmids containing the gene of interest Protease K (BoehringerMannheim) NaCl and lmmol/L EDTA TE [lOmmol/L Tris-HCl (pH 8. 0 ) and lmmol/L EDTA TE equilibrated with phenol: chloroform: isoentyl alcohol (25 : 24 : 1, V/V/V) Virus: T0 Z.1 virus (K riskyetal.1997, 1998b) X-gal color solution 300ug X-gal/ml TBS 14 mmol/L K4Fe(CN)6O 14 mmol/L K3Fe(CN)6O X-gal is insoluble in water and must be dissolved in dimethylformamide before addition to the color solution. Cell Scraper Centrifuge bottle (500 ml polypropylene) C O 2 Incubator Cup-horn Ultrasonic Crusher Thermos bottle (T-75 cm2) Nozzle Nutator Rotary Table Phase L o c k Gel H e a v y Tubes (Eppendorf) Culture plates (6-, 12-, and 96-well plates) Preparative centrifuge with J L A 10.5 head (Beckman) Roller bottle (850 cm2) Syringe with needle (3m l ) Tube: 13 ml B e c k m a n 17m m path-length seal 15 ml conical flask Ultracentrifuge with N V T 65 turntable (B e c k m a n X L -90) Wide Mouth Head Methods 1 - 2.4X 107pfu of TOZ.1 virus was added to approximately 8 X 106 auxiliary 7b cells (infected plural = 3). 2 . Transfer the infected cells into T-75 bottles. Incubate at 37 °C in a CO2 incubator for 18 to 24 h. All cells should be infected with virus and adsorbed on the bottle. 3. Scrape the cells with a cell scraper. Transfer to a 15m l conical tube. Centrifuge at 2060 g at 4.4 °C for l Omin. remove the supernatant. 5- Add I ml of lysis buffer with 0. l m g/m l of Proteinase K to the tube. 6 . Place the tube on a N u ta to r rotating table at 37° C overnight. 7 - Transfer the solution to a P h ase L ock G el H eavy tube. 8- Add Im l of phenol: chloroform: isoamyl alcohol (25 : 24 : 1). Mix gently for 1 to 2 min. 9.3020 g ■ Centrifuge for 5 m i n . 10.Transfer the aqueous phase to a new tube. 11. Add 2x volume of isopropanol. Mix well. 12. Wrap the precipitated DNA (white flocculent) around the glass tip. 1 3 . Transfer the draped D N A to a new tube. Wash with 70 % ethanol. Allow the D N A to air dry. 14. Add 0.5 ml of T E buffer to the dried tube. Incubate overnight at 25°C. 15. Resuspend DNA by blowing gently with a wide-mouth tip. The use of a wide-mouth tip (Gilson) minimizes the breakage of viral DNA and thus increases the infectivity of viral DNA. 16 - Determine the concentration of D N A (OD260) using a spectrophotometer. 17- The gene of interest was cloned into U L 41 shuttle plasmid. 18-The day before transfection, 5X 105 7b cells were seeded into 6-well plates containing D M E M /10% F B S . 19- Cut viral DNA (obtained from step 15) with PACI for 6 h at 37°C according to the manufacturer's method. 20- Transfect viral D N A and plasmid D N A into cells with Lipofectamine 2 0 0 0 according to the manufacturer's method. Plasmid vectors should be linearized prior to transfection to improve the efficiency of recombination compared to unenzymatically cleaved superhelical plasmids. Enzymatic cleavage of the plasmid to release the insert fragment and purification of the restriction enzyme fragment does not improve recombination efficiency. However, the use of purified fragments is recommended because it reduces the possibility of inserting the plasmid vector sequence into the virus due to hemizygous recombination. 2 1 . Add fresh DMEM/10 % FBS and incubate at 37°C. 22- After plaque formation, harvest the medium and cells. 23- Repeat freezing and thawing of cells 3 times. Ultrasound breaks the cells. Centrifuge at 2060 g at 24.4°C for 5 millimeters to remove cell debris. Mix the supernatant with the medium obtained previously (step 22). Store the supernatant at 80°C for storage. 25. Determine the titer of the stored recombinant virus. a- The virus was subjected to a series of 1 ○-fold dilutions (ICT2 ~ 1 ○_1°) with serum-free medium. b- Add l ○○ Ml of virus per dilution to a 12-well plate containing 4X105 7 b cells/well. c. Incubate the plates for l h at 37°C in a C O 2 incubator. Add I ml of DMEM/10 % FBS and incubate overnight in the incubator. d- For the next 24 h, remove the medium. Cover the monolayer of cells with I m l of 1 % methylcellulose/10 % F B S solution. The e- Incubate the plate for 3 to 5d until plaque formation. f. Take out the methyl fiber-based membrane and stain it with crystal violet solution for 5m i n . g- Perform a spot count and count the number of original pfu/l m l . 26- Add 30 p f u of virus to a number of I m l suspension cells of approximately 2X 106 placed in I5m I conical tubes. The tubes are placed on a Nutator rotating table for I.5 h at 37° C. The tubes are placed on a Nutator rotating table for I.5 h at 37° C. 27- Add 9m l of fresh medium. Inoculate 100ul per well in a 96-well plate. 28- Incubate the plates in a C O 2 incubator at 37°C for 3 to 5d until plaque formation. 29- X -gal staining was used to determine which samples contained the plaques formed by the desired recombinant virus. a. Transfer the medium from the 96-well plate to a new dowell plate. Store this plate at 80°C for the next round of restriction dilutions. b- In a 96-well plate with transfected cells, add 100/J of X -gal staining solution per well. c. Incubate the plate at 37°C for 1 to 18 h until blue patches form. d. Identify wells that contain only one white patch. If the gene of interest in the viral recombinant replaces ZacZ in T O Z .1, a white patch will form. 3 ○ - Perform two more rounds of restriction dilution spot isolation with virus stored at 1 80° C (steps 26 to 29). 3 1 . Isolate the virus DNA and use Southern blotting to determine the presence of inserted genes and the absence of deleted genes (step 29a). At this step, the preserved virus can be used to prepare high-titer stock solutions required for general experiments. 32. In two 850c m 2 roller flasks, each spiked with I O O m l containing approximately 2 X I O 7 helper cells of D M E M /10%. FBS medium. Incubate at 37°C. If not incubated in a CO2 incubator, add 1.7 ml of HEPES (pH 7.35) to each bottle to adjust the medium. buffer. 33. When the cell monolayer reaches approximately 75% confluence (2 to 3 d), aspirate the medium. Add 100 ml of serum-free DME containing 0.02 to 0.05 viral multiplicity of infection. Incubate at 37°C for I.5 hours. 34. At the end of the uptake phase, add 90 ml of DME/10% FBS containing Im Hepes. Incubate at 37°C for 2-3d. Observe the bottles with a light microscope to determine the optimal time to harvest the virus. The time varies according to the different viral backbones . In general, the cytotoxicity of the virus is evident when most of the cells have undergone curling, but have not yet been dislodged from the surface of the bottle. 35. Use a cell scraper to scrape the cells into the culture medium. 36-Harvest the medium containing the suspended cells. Centrifuge at 2060 g for lOmin at 4°C. 37- Transfer the supernatant to a 500 ml polypropylene centrifuge bottle. Place on ice. 38.Resuspend cells with a minimum volume (2 to 5m l ) of serum-free D M E D . Crush with three rounds of ultrasound to release cell-associated virus. 39. Centrifuge at 2060 g for l Omin at 4°C. Remove supernatant. Mix with supernatant from step 37. Place on ice. 40 - Centrifuge the mixed supernatant at 18,600 g for 1 h in a BECKMANN preparatory centrifuge with a J L A 10.5 turntable. 41. Discard the supernatant. Add 500 ul of PBS to each roller bottle containing virus-infected cells and resuspend the virus with sufficient shaking. 42. Transfer the supernatant to a 13 ml BECKMANN 17 _ path-length closed tube. Add 20 % iodixanol until the total volume is 13 ml. Close the tube. 43. Centrifuge for 4.5 h at 342,000 g in a BECKMANN XL-90 ultracentrifuge with a NVT 65 turntable. 44. Insert a 3 ml syringe needle into the closed tube and aspirate 1 to 1.5 ml of virus (upper) strip (Figure 2). Almost 60% of the virus will appear on the second band from the bottom of the gradient. Sometimes a higher defective band may appear when preparing with wild type. If there are three bands, take the middle one. Alternatively, the tubes can be fractionated and determined to determine the titer of each fraction 45-integer-fold dilution of the virus and determine the titer. Store the virus at 80°C. Optiprep can be used as an antifreeze. Optiprep can be used as an antifreeze. a low infection multiplicity of 3 to 5 is sufficient to transduce 100% of cells, unlike other viruses such as adenoviruses, AAVs, standard retroviruses and lentiviruses. The virus can be transduced in vivo by direct injection at various doses. For more product details, please visit Aladdin Scientific website.
Tris salt buffer (TBS) (pH 7. 5) [50 mmol/L Tris-HCl (pH 7. 5) , 150 mmol/L
Apparatus
Rotate the culture at 37°C for lh.
Depending on the virus, plaques are formed within 3~5d.
If recombination has occurred, the gene of interest replaces the reporter gene.
Preparation and Purification of Virus Storage Solution The following steps are used to prepare the virus storage solution using two roller bottles of cells. Please adjust the scale as needed.