Generation of VSV-G-pseudotyped retroviral vectors
Generation of VSV-G-pseudotyped retroviral vectors
In order to improve the efficiency of transduction, a strategy called pseudotyping, which combines retroviral particles with other virally encoded capsid proteins, has been adopted. Because these pseudotyped vector particles use a different surface receptor to enter the cell, they can transport the transgene into mammalian cells more efficiently than vectors containing the original retroviral capsid protein. If the receptors have a strict tissue distribution, then relying on the capsid proteins, they are also capable of tissue-specific transport. Here, we used the glycoproteins (G ) of vesicular stomatitis virus (V S V ) to illustrate how pseudotyped vectors can be generated within a few days. Pseudotyped lentiviral vectors were generated by a similar strategy. Author: T. Friedman et al, Translated by W. Qin et al. This experiment is from "Gene Transfer".
Operation method
Generation of VSV-O pseudotyped retroviral vectors Move Generation of VSV-O pseudotyped retroviral vectors Material reagents CaCl2 2mol/L Dissolve 29. 41 g of CaCl2 - 2 H20 into 100 ml H2O, filter aseptically. Cells 293T (ATCC: CRL-11268) HT1080 (ATCC: CRL-121) DMEM containing 4.5 g/L glucose, 3.7 g/L NaHCO3, 2 mmol/L L-glutamine, 100 mg/L gentamicin, and 2 mmol/L L-glutamine. Ethanol (100%) Fetal Bovine Serum (FBS) 2X HBS Dissolve 2. 38 g of HEPES, 3. 28 g of NaCl, 42. 4 mg of Na2 HPO4 in 180 ml of H2O, adjust pH to 7.12, add water to 200 ml, and filter aseptically. Lactose solution (4 %) Dissolve 4 g of a-lactose monohydrate (SigmaAldrich) in IOOml PBS and filter aseptically. P E G 8000 ( 40 %; Sigma-Aldrich ) Dissolve 4Og of PEG in 100 ml of PBS, stir overnight at room temperature, then autoclave and store at 4°C. Phosphate Buffer Solution (PBS) Dissolve 8 g NaCl 0.2 g KC1, L 44 g Na2 HPO4, 0.24 g KH2PO4 in 800 ml H2O, adjust pH to 7.4, make up H2O to 1L, autoclave. 4, replenish H2O to 1L, and autoclave. Plasmid Plasmid: pCM V -G P V SV-G Expression Plasmid: PCMV -G Polyglutamine (4mg/ml in PBS; Sigma-Aldrich) Reverse transcription virus carriers containing GFP genes or drug screen markers lm m o l/L T ris (p H 7. 9) 0.lm m o l/L E D T A Sterile filtration. Instrumentation A c ro d isc injector filters Use 0.45um low protein binding filters (Pall Gelman laboratory, AimArbor, Michigan). A centrifuge (B eckm an Allegra 6 tab leto p ) with a PTS-2000 rotor was used. Petri dish (100 m m ) E ppendorf thermomixer R m ixer (E p p en d o rf, W e stb u ry , N ew Y ork ) Flow Cytometer (FACS) Syringe (IOml) DNA Transfection Tubes (12m m X 75m m polystyrene) Centrifuge tubes (Ultracentrifuge, 25m m X 89m m; Utra-Clear, B eckm an C oulter) Ultracentrifuge (B eckm an C oulter) SW 28 rotor head METHODS Carrier generation 1 . Inoculate 2 x 106 293T cells on a 1,000 mm Petri dish, complete with DMEM/1 0 % FBS, and incubate cells overnight. The passaging time of 293T cells was about 20 h, and this cell line was able to maintain a high transfection efficiency within 2 months. However, the cells suddenly lose the ability to produce vectors after 3 months of growth. Therefore, cells should not be cultured for more than 3 months. 2- Replace the old medium with IO ml of fresh medium and continue to incubate the cells in CO2 incubator for 1 to 2 hours. 293T cells proliferated rapidly. Change the medium on the day of transfection to ensure that the medium has optimal p H for effective transfection. Cell density should be 7 0 % confluent at the time of transfection. Low cell densities will reduce vector titers. Transfection at 80 % confluent cell density has been shown to produce high titers of vector. Higher cell densities not only increase vector yield, but also extend the time period for vector collection, as the cells are better able to resist VSV-G-induced cell separation at higher densities. 3. Prepare fresh transfection solution as follows: b. Add TE79/10 solution to a total volume of 437 m1 . c. Force [I 63pl 2mol/L CaCl2, mix. d. Add 500M 12XHBS solution drop by drop with constant stirring. e-Incubate at room temperature for 30 min to allow calcium phosphate and DNA to form a precipitate. After 30 min of incubation, a calcium phosphate precipitate should form and the solution should become turbid. If not observed, repeat the precipitation step with fresh transfection solution. 2 X H B S The p H of the solution is the most critical factor for successful transfection. If the p H of the solution changes during storage, it should be replaced with a fresh solution. V S V - G is toxic to mammalian cells and increasing the amount of p CM V - G transfected will result in a decrease in vector titer. This can cause transfected 293 T cells to die before maturation due to overexpression of VSV-G. Therefore, the amount of p C M V - G should be kept to a minimum during vector production. 4 . Add the DNA precipitate to the 293T cells prepared in steps 1 and 2, mix well, and incubate on a petri dish for another 5 to 6 h. Replace with 6 ml of fresh medium and incubate overnight. The reason for using 6 ml of medium (instead of IOml) is to reduce the volume of the collected vector for the following vector purification and concentration steps. 5. Collect vector-containing medium from the same petri dish 24, 40, 48, 64 and 72 h after DNA transfection. For each collection, replace the medium with 6 ml of fresh medium for the next vector collection. The cytotoxicity induced by VSV-G overexpression becomes increasingly obvious. At 72 h post-transfection, most cells are detached from the culture dish. If no cytotoxicity such as siderophore formation or cell detachment is observed at this time point, the transfection may have failed and a low vector titer can be expected. 6- Centrifuge the medium in a PTS-2000 turntable, Beckman Allegra 6 tabletop centrifuge at room temperature 2500 r/min for 5 min. collect the supernatant and filter it through a 0.4 5 um syringe. The supernatant was collected and filtered through a 0.45 um syringe. The medium is immediately stored at 80°C until the purification step. Vector purification 7. Dissolve the crude carrier preparation and mix with 40 % PEG reservoir to achieve a final PEG concentration of 10 %. 8. Place the PEG carrier mixture on ice for 4 h or refrigerate overnight. The carrier in PEG is very stable: it can be stored at 4°C for 1 week with minimal loss of infection titer. 9- Centrifuge the vector at 3000r/min in a PTS^2000 rotor head, Beckman Allegra 6 tabletop centrifuge, for 3Omin0. 10. Resuspend the vector with 4 % PBS with lactose or 10 % FBS with DMEM. Collect the resuspended vector in 1/10 or 1/30 of the original volume. Partially purified vector can be used directly for transduction or continue ultracentrifugation to obtain higher titers of vector preparation. The addition of lactose or FB S to the resuspension solution stabilizes the M LV vector when stored. Solutions containing resuspended vectors are usually turbid because of the presence of residual PEG, and increasing the volume of the resuspension solution reduces the turbidity. However, the residual PEG does not affect cell transduction. Residual PEG in storage appears to stabilize the vector. Prolonged storage at 80°C has little or no loss of infectivity, and multiple freeze-thawing has a much lesser effect on vector titer than vectors that have not been treated with PEG. Gel electrophoresis and silver staining of vector preparations before and after PEG purification revealed that PEG precipitation removed a significant amount of contaminating cellular proteins. In terms of band concentration, more than 95% of the FBS was removed by this step. In terms of titer, the plant recovery in this step was high (80% to 100%). Vector Concentration 11 . The Ultra-Clare ultracentrifuge tubes (25 m m X 89 mm), adapters, and the screw cap of the SW 28 rotor head are sterilized with 100 % ethanol and air-dried in a laminar flow hood. 12- Transfer the P E G purified carrier preparation to a sterile ultracentrifuge tube and add P B S or D M E M to 37 ml. 13- Centrifuge the carrier in a S W 2 8 rotor, B e c k m a n C o u l t e r ultracentrifuge at 4°C , 25 O O O r / m i n 90m i n 14. Discard the supernatant and invert the tube in a laminar flow hood for a few minutes to remove the liquid. Resuspend the carrier with 0.5 ~ I m l 4 % lactose or DMEM. 15.4°C, oscillate the carrier in lactose solution or DMEM in an Eppendorf thermomixer R mixer at 800r/m i n for 3 to 4 h . The resuspended vector solution is turbid because of residual PEG. If the vector is resuspended in DMEM, add 10% FBS and store the vector preparation at 130°C. Titer Determination 16.H T1080 were inoculated at 2X106 on 100 mm dishes with complete medium D M E M /l ○%F B S . An additional Petri dish was prepared for cell counting the next day. Cells were cultured overnight. 17.- One dish is digested and counted, and the other replaces the old medium with fresh medium containing A fjtgA n l of polyglutamine. Dilute the concentrated medium with DMEM, add to H T 1080±§ dishes, and incubate overnight. 18. Replace with fresh polyglutamine-free medium and continue incubation overnight. 19-Digest cells, centrifuge, and resuspend with l m l D M E M /10% F B S for flow-through analysis. 20-Calculate carrier titer (infectious particles/m l ). For more product details, please visit Aladdin Scientific website.
T E 79/10
a. Mix 15ug of p P Y -1, 15M g of p C M V -G P , 2ug of p C M V -G in a 12 mmX 75xnm polystyrene tube.
Fresh medium should be added carefully along the edge of the dish, as the toxicity of transfection and VSV-G expression can cause cells to dislodge easily.
The collected vectors can be directly ultracentrifuged without PEG purification. However, the resulting vectors are more likely to cause cytotoxicity in different cell types than PEG-purified vectors. Therefore, the PEG procedure is highly recommended.
Total number of cells on the day of transduction X Percentage of G F P + cells as determined by F A C S X Vector dilution factor
If drug-selective labeling was used in the titer determination, 2 X IO5 H T 1 0 8 0 cells per I O O m m culture dish, since selectivity is most effective when cells are actively proliferating. If cells are confluent in the culture dish, other selections are needed to eliminate the background of nontransduction:.