Splenic necrosis virus-based vectors
Splenic necrosis virus-based vectors
The experimental approach described in this chapter is based on our experiments with carriers from S N V and its closely related R E V -A (P a r v e
e n e t a L 2000). We have used both viral vectors for gene transfer to a variety of cells, including initial hematopoietic cells, human brain, and postmitotic neuronal cells (Parveen etal. 2000). As with other retroviral vectors, the three major components of the SNV vector-based system are (i) the packaging signal sequence (shame, to ensure that the vector RNA is shelled into the retrotransposable particles; (ii) the retrotranscriptional machinery; and (iii) the sequences necessary for the entry of the pre-integrating viral vector into the host cell (Kurian etal. 2000; Dornbrug 2003). To enhance the therapeutic potential of viral particles, the viral life cycle is genetically modified, i.e., these particles can transfer only one therapeutic gene upon transfection without further replication.
Author: T. Friedman et al., Translated by W. Qin et al. This experiment is from "Gene Transfer".
Operation method
Generation of retroviruses based on the S N V vector system and their application in transduction of various cell types MATERIALS Move Generation of S N V vector-based retroviruses and their use in transduction of various cell types MATERIALS reagents Calcium chloride (CaCl2) (2mol/L) Dissolve 14. 7 g CaCl2 in IOOml H2O. Decontaminate by filtration or autoclave. Transfected cell lines: 293T and D17 (ATCC) Glycerol (10 % ~ 20 %) (suitable) H ank balanced salt solution (HBSS) 2 X H E P E Buffer (HBS: 280m m ol/LN aCl, 10 m m o l/L K C l , 1.5Imnol/LNa2 H P 0 4< ! > , 12 m m o l/L glucose, 50 m m o l/L H EPES, pH 7.5). LacZ colorant (Chemicon) Culture media Complete medium: DMEM medium containing 10 % Fetal bovine serum F CS (293T cells) or F B S (D 17 cells), 1 % Penicillin, 1 % Glutamic Acid. Serum-free medium with 1 % penicillin, 1 % glutamic acid Phosphate buffer (P B S ) (suitable, - required if manipulating glycerol shock) Nuclease-free water Plasmids for generating retroviruses Plasmids encoding viral envelope proteins Gene transfer vectors containing the target gene Plasmids encoding E n v proteins SNV- and REV-A-based viruses are pseudotyped to express VSV-G or other Env-encapsulated proteins that specifically target ligands for human cell surface receptors. The quality of the plasmid DNA used for transfection is critical for generating high titer retroviral vectors. It should be ultrapure and should avoid any chemical contaminants, RNA or proteins. We recommend that the purified DNA used for retroviral particle generation have an A2m:A28 value greater than 1.8. Polyglutamine Transfenctam reagent (P r o m e g a ) (suitable) Instrumentation Cell culture dishes [IOO m m (or 12-well or 2-4-well plates for multiple transfection) and 6-well plates]. Centrifuge tubes (15 ml) Filter: Acetic acid or polysulfonated cellulose (binds small proteins), 〇.45um membrane Incubator, preset to 37°C Small centrifuge tubes (1.5 m l, sterilized) Microscope Pipette (1.0m l or Pasteur pipette) Shaker Ultracentrifuge (B e c k m a n ), S W 41 turntable Methods Preparation of cells 1. 24 h before transfection, grow 293T or D 17 cells in complete medium in IOOmm plates overnight. The planting density depends on the cell proliferation ability. Generally speaking, IOOmm plates are planted with 0.5 X IO6 ~ 0.8 X 106. 2. Observe the cell density, which should be 75% to 80% confluent. Replace the original medium with IO ml of fresh complete medium. Incubate for 4--6 h. Cellular confluence plays a major role in obtaining optimal viral titers. Transfection This method is suitable for IOOmm cell culture plates. The amount of D N A transfected should vary with the size of the plate used for transfection. 3. For each I O O m m plate, add 0 -5 m l of 2 X H E P E S buffer to a sterilized I.5 m l small centrifuge tube labeled Tube 1. For multiple transfections, it is simpler to prepare the transfection mixture in 12 or 2 4-well plates. 4. Another tube, labeled as tube 2, is filled with IOug of Gag-Poel Coating Plasmid, 4 to 5 cups of Env (V SVG or SNV wild type) Coating Plasmid, and 10 hong of Transfection Vector. The total amount of plasmid DNA should not exceed 30 ug. Add 62ul 2m ol/LCaCl2 and sufficient nuclease-free water to bring the total volume to ○ . 5 m l . 5 . Add the mixture in Tube 2 dropwise to Tube 1 with an I.O ml pipette or Pasteur pipette and suck vigorously, or shake Tube 1 during addition. Alternatively, oscillate tube 1 during addition of tube 2. The final solution will produce a precipitate, which is milky white in color. 7. Blow up and down with a pipette or oscillate to mix the precipitate well. 8 . Add I m l of suspension and 5 m l of serum-free medium to plates containing 2 9 3 T or D 1 7 cells. Slowly add the mixed suspension drop by drop and vortex gently. 9. Incubate at 37°C for 4 to 6 h. Incubate for 2 hours. Replace the original medium with fresh medium containing 100 % FCS. Incubate for 36~40 h to produce recombinant virus. Glycerol shock (if appropriate) Some laboratories perform glycerol shock at this step to increase the uptake of conductive D N A by the nucleus. In our basal 10 - If glycerol shock is chosen, proceed as follows: a- Aspirate the medium and wash twice with 5 ml PB S or medium. Shake the plate during the wash and microscopically b. Add 2 m l of glycerol of the determined concentration to the cells for 2 to 4 min. c- Discard glycerol, PB S wash, and add IO m l of fresh medium. Collection of retroviral vector supernatant 11. For viruses generated based on SN V vectors, collect the supernatant from transfected 293T or D 17 cells 36~40 h after changing the culture medium. The supernatant of transfected 293T or D 17 cells was collected after 36~40 h of culture medium change. The supernatant was centrifuged at 1500r/m in a 15-mi tube for 5 min, and the cell debris was discarded. 12-Transfer supernatant. Use immediately for titer assay or freeze at _80 °C until needed for transfection. For S N V and R E V - A based vector systems, the biological titer is used as a measure of virus yield. Care should be taken with supernatants to avoid multiple freeze-thawing as this can result in a severe reduction in reverse transcription virus titer. Concentration of retroviral particles 13. Filter the retroviral vector supernatant through a 0.45um membrane. Do not use digestive fiber membranes because proteins can bind to the membrane and destroy the surface of the viral membrane. 14. H Bekman Ultracentrifuge SW4 Turning Head, 4°C, 25 000r/m in Centrifuge 2 h Supernatant. 15. Slowly discard the supernatant without stirring the precipitate. Resuspend the precipitate containing retroviral particles with 50 f J H B S S . 16-Determine the titer of the concentrated retrovirus as described in the following section, or freeze at 80°C until next application. Since freezing and thawing significantly reduces the titer, especially for SNV, it is best to prepare it now. Determining transduction efficiency of retroviruses This process requires that either the virus or other reporter genes be integrated into the transfer vector. 17 - Determine the retroviral titer as follows: For adherent cells b . Discard the medium and add 0.1 ml of virus carrier supernatant containing reverse transcribed virus particles and polyglutamine at a concentration of 100 ug/ml. Alternatively, Transfectam reagent is used at a concentration of 2ug/ml. Transfectam reagent has been observed to be less toxic than polyglutamine. c. After 3 to 5 h (depending on the cell type), replace the virus-containing medium with IO ml of fresh medium. Continue to incubate the cells for 48 h. d. If any of the reported genes are present, the blue cells are stained with IacZ reagent and counted to express the titer in clone forming units (cfu)/ml. If other reported genes are used, immunocytochemistry or real-time polymerase chain reaction (PCR) can be used to accurately detect gene expression and viral titer. For transduction of non-mounted cells (suspension cells) a-Target cells were grown in 6-well plates at a density of 1. 5x106 cells/m l of medium for 12~18 h prior to transfection. b. Centrifuge cells in suspension. Resuspend cells in virus carrier suspension at a density of 1.5X 106 cells/m l in the presence of polyglutamine (8ug/m l) or Transfectam (2ug/m l). As mentioned earlier, T r a n s f e c t a m is a better choice due to its lower toxicity. c. If polyglutamine is used as a transfection reagent, it is necessary to observe its effect on cell proliferation and growth. d. After 4~5 h, centrifuge to discard viral particles and incubate in complete medium for 48 h as described for adherent cells. e. To further increase the efficiency, multiple transfections can be performed 12 to 24 h after the initial transfection. f. Fix and count the transfected cells to determine cfu/ml. Post-mitotic neuronal cells were transduced with SNV-based retroviral particles. This method was used to transfer the Z a c Z reporter gene to postmitotic differentiated neuronal cells. a. Postmitotic differentiated neuronal cells were prepared according to the procedure previously described by M u k h t a r et al. b. Cover cells with a mixture of reverse transcription virus microparticle suspension and T r a n s f e c t a m reagent (2ug/m l ) . Leave for 2 h. c. Repeat the retroviral particulate coverage procedure 3 times at 24-h intervals. d. Determine the expression of reporter or therapeutic genes in these post mitotic cells by complementation techniques. For more product details, please visit Aladdin Scientific website.
The amount of D N A transfected should vary with the size of the plate used for transfection. Reagents (CaCl2, 2 X H E P E buffer, nuclease-free water) can be homemade or purchased (e.g., Prom e g a's ProFection M a m m a l i a n Transfection S y s t e m - C a P O 4 )
6 . Leave the mixture at room temperature for 20 to 30m in .
experiments based on the generation of S N V retroviral particles, there was no great difference between treating the cells with glycerol shock or not.
observation to ensure that most of the precipitate is removed. Repeat the wash if there is still a lot of precipitate.
The percentage of glycerol, from 10% to 20%, should be determined empirically. In general, a higher percentage of glycerol is used if it has no effect on the proliferative capacity of the cells.
a-planted rake cells D17 (for determining the titer of SNV wild-type encapsulated pseudotypes) or 293 T, complete medium, density less than IXIO6/IOOmm dish. Grow for 24 h.
Half-dose transfection is performed 5 to 6 h after virus infection of cells. Overnight, the cells are transfected at the maximum dose.
This is because suspension cells are somewhat more sensitive to toxic effects compared to adherent cells. Shake on a shaker
Plate. For SNV, to produce optimal transfection efficiency, the concentration of polyglutamine is at 8 ~ SOug/ml.