Lentiviral vectors can be designed as replication-incomplete or replication-condition-dependent (motile) vectors. HIV-2 vectors are more effective in gene therapy against H IV because they are less pathogenic than the corresponding HIV-I vectors and are less likely to recombine with HIV-I in infected cells. This chapter discusses the design of the HIV-2 vector, its application, and its use as a vector in trials for the transport of genes of interest, particularly in anti-H IV gene therapy. Author: T. Friedman et al, Translated by W. Qin et al. This experiment is from "Gene Transfer".
Operation method
Generation of HIV-2 vector particles by gene transfer Move Generation of HIV-2 vector particles by gene transfer Materials reagents CaCl2 (lm ol/L ) Transformed and transduced cell lines: 293 T cells or 293 F T human embryonic kidney cells Produces DNA for H IV-2 carrier particles HIV-2 Vector DNA Package carrier (HIV-1, HIV-2, or FIV) Membrane Plasmid (VSV-G) DMEM culture with 10% fetal bovine serum FBS, amino acids, antibiotics, antimicrobials. Ig HEPES acid (Sigma-Aldrich H3375) 1.6 g NaCl 0.72 ml 0. 25mol/L Na2 HP04 Iml lm ol/L KCl was added to 100 ml of double distilled water and the pH was adjusted precisely to 7.5 mg/L with NaOH (5 mol/L and 1 mol/L). 12 PBS containing 2 % polyformaldehyde PBS Polyglutamine storage solution (bemecium bromide; 8m g/m l dissolved in P B S) Trypsin Equipment Flow Cytometer Fluorescence Microscope rotating head 4 5 T i Fixed angle swivel head (Beckman-Coulter) 45Ti Leveling Head (Beckman-Coulter) Standard tissue culture equipment, including 100 mm diameter tissue culture dishes and 12-well plates Filters (0.45um pore size) Benchtop centrifuge Methods HIV-2 carrier production (calcium phosphate method) 1. One day before transfection, inoculate about 4. 0x106 293 D cells or 293 FT human embryonic kidney cells into a 100mm diameter tissue culture dish, add 8~IOml of DMEM medium containing 10% FBS, amino acids, and antioxidants (if necessary), and incubate overnight at 37°C in a 5 % CO2 incubator. 2. Change the medium 1~2 h before transfection. 3 . Prepare phosphate-depleted-DNA coprecipitation. Mix 15ug of HIV-2 vector DNA, IOug of packaging vector (HIV-1, HIV-2 or FIV) and 5 clusters of VSV-G plasmid, then add 374ul of sterile water and 126ul of 1mol/L CaCl2 to the plasmid mixture. 4. Take the spread 293T host cells from the incubator to the tissue culture bench. 5. While mixing the plasmid mixture in Step 3, pipette 500 ul of 2X H B S dropwise with an Im l pipette. Use the same pipette to add the calcium phosphate-DNA suspension drop by drop to the host cells. Then put back into the incubator to continue incubation. 6. After 6~8 hours of incubation, gently remove the medium from the petri dish and replace it with 10 ml of fresh medium. Collect the carrier pellet 7. After 2d of transfection, remove the supernatant from the medium and put it into a 50m l conical tube and replace it with fresh medium. Store the supernatant at 4°C. 8. After 3d of transfection, collect the supernatant again and put it into the previous sample stored at 4°C. If the cells are still alive, add fresh medium, 24 ml of fresh medium, and then add the supernatant to the sample. If the cells are still alive, add fresh medium and repeat the process 24 h later. 9. Centrifuge for 15 min at 2800r/miri in a benchtop centrifuge to remove unwanted cell debris. Transfer the supernatant to a new centrifuge tube and filter through a 30 ml syringe and a 0.45 JUII1 pore size filter. Store the filtered supernatant in 2m l aliquots at 80°C or concentrate as described in steps 10 to 12! Storing the filtered supernatant in aliquots avoids freezing and thawing of all the stored carriers during titer determination. Carrier Concentration 10. Centrifuge the filtered supernatant containing the vector at 42,000 max (19,000r/min in a fixed-angle rotor 45 Ti) for 2 hours. 1 1 . Remove the supernatant, invert the tube onto a sterile gauze and dry for 2 min. 12. Dry the walls of the tube with sterile gauze, then resuspend with 20-50 ul of IXPBS, mix all aliquots together, and store at 80°C until needed. The vectors can be stored for up to one year; longer storage will reduce the titer of the vectors. Virus titer assay Viral titer can be measured using various cell types. This formula assumes that 2 93T cells are used for the assay and that green fluorescent protein (GFP) expression is used as a marker. 13. The day before the viral titer is measured, 293T cells are inoculated into a 12-well plate at a density of 2X 104 to 4 X 104 cells/well. Incubate overnight at 37°C in a 5 % CO2 incubator. 14. On the day of the titer assay, prepare a series of LOml carrier culture medium dilutions (5 to 10 times the spacing) ranging from IO2 to IO6 particles/ml and containing 8ug/m l of 1,5-dimethyl-1,5-diazoundecanoylmethylene polybromide (Polybrene). The number of cells in each of the 12 wells was counted and repeated 3 times to obtain the number of cells before transduction. Add the diluted vector to the relevant wells and incubate overnight. The expected titer is approximately IO6 transfection units/ml. 15. The next day, aspirate the medium and replace it with fresh medium. Continue culturing the cells for 2~4d. 16. Remove the medium, add 500ul of trypsin and incubate at room temperature for 1~2 min. remove the trypsin, gently knock the cells loose and collect the cells with Iml PBS. Fix the sample and negative control with PBS containing 2 % paraformaldehyde. GFP expression in the samples was detected by flow cytometry. 17. Plot the number of viral particles per well against the percentage of GFP-expressing cells to determine the dilution required when 50 % of the cells are expressing GFP, and calculate the number of transfection units/mi in the vector reservoir from this. Another formula for calculating vector titer is TU/ml= ( % G FP positive cells/ioo) X (number of transduced cells) X (dilution factor) 18. The day before transduction, inoculate adherent cells at a density of approximately IXlO5 cells/well in a 6-well plate or 4 X 106 cells in an IOOmm dish. Incubate overnight in a 37°C, 5% C02 incubator. 19. On the day of transduction, remove the medium. Add 2 ml of FBS-free medium, 1 ml of freshly thawed carrier storage solution, and 8ug/ml final concentration of polyglutamine. Incubate in a 37℃, 5% CO2 incubator for 2 hours. 20. Aspirate off the supernatant and add fresh medium to the cells. Place the cells in a 37℃, 5 % CO2 incubator for 2~4d, and analyze the GFP expression by flow cytometry or sudden light microscopy. Transduction of Suspension Cells 21. Collect suspension cells, count, and centrifuge at 500 g for 5m in with I 5m I polypropylene tubes to collect 3 X 106 ~ 5 X 106 cells. 2 2 . Suspend the cell starch with I ml of FBS-free medium. 23. Add I ml of freshly thawed carrier storage solution and mix gently. 24. Add condamine to achieve a final concentration of 8ug/ml. 25. Centrifuge the cell/carrier/polyglutamine mixture at 10000 g for 2 h at room temperature. 26. Aspirate off the supernatant and resuspend the cells with fresh medium. Place the cells in a 3700 c, 5% CO2 incubator for 2~4d and analyze the G FP expression by flow cytometry or fluorescence microscopy. For more product details, please visit Aladdin Scientific website.
2X HBS
Transduction of adherent cells