This chapter describes how to construct H S V /A A V heterotrimeric vectors using an unassisted viral packaging system, and also discusses its advantages and disadvantages compared with conventional H V S /AAV amplicon vectors. Author: T. Friedman et al, Translated by W. Qin et al. This experiment is from "Gene Transfer".
Operation method
Construction and packaging of HSV/A A V hybrid amplicon vectors Move Construction and packaging of HSV/A A V hybrid amplicon vectors Materials reagents Cells: V e r o cells (clone 76, E C A C C 85020205) and V E R O 2-2 cells (Smith etal. 1992). Dry ice DMEM (Invitrogen) Ethanol Fetal Bovine Serum (FBS 2%, 6% or 10%) G418 (Geneticin; Invitrogen, 11811-031) H A N K Buffer Liquid (H B S S ; G I B C O B R L ) Lipofectamine reagent (Invitrogen) Plasmid HSV/AAV hybrid amplicon plasmid (pHSV/AAV; H eisteretaL 2002) Packaging-defective H SV -I helper virus DNA (Saeki et al. 2001) fHSVApacA27AKn pEBHICP27 Opti-MEMl (Invitrogen) PBS buffer Sucrose Membrane Enzyme (0.25%), EDTA (〇 _02%) (Invitrogen) Instrumentation Disposable syringe (20 ml) Blood Counting Plate Incubator (humidified, 37°C, 5 % CO2) Ultrasonic crusher with probe only (e.g. Sonifer 250; Branson) Syringe needle filters (〇.45 mm, polyethersulfone membrane; Sarstedt) Tissue culture flasks (75 cm2) Tissue culture plates (60 mm diameter) Ultracentrifuge (Sorvall SS--34 head) Ultrapure centrifuge tube (30 ml, 25 mmX89 mm; Beckman) Methods Co-transfection of packaging-defective HSV-I helper virus DNA with vector DNA 1. Preparation of V E R 02-2 cells to be transfected a. Cultivate VER02-2 cells with DMEM medium containing 500 ptg/ml G418 and 10% FBS for two passages per week, and place the cells at a ratio of 1 : 5 in 75c m 2 tissue culture flasks in 15m ! Fresh medium was used for passaging. b. On the day before transfection, remove the medium and rinse the cells twice with P B S. The cells were then washed with P B S. The cells were then washed with P B S. c- Add 3 m l of trypsin/E D T A . Incubate the culture flask at 37°C for l O min to deliquesce the cells. d-Cells were counted using a hemocyte counting plate. Each 60 m m diameter plate was inoculated with I.2 X I O 6 cells in 3 m l of D M E M medium with 10% F B S . 2- Prepare two 15m l conical tubes filled with 2504 O p t i - M E M I for each 60m m diameter plate. a- Add 0 -5ug of P H S V /A A V , 2 ug of f H S V A p a c A 27A K n , and ○.2ug of p E B H I C P 27 to one of the conical tubes. mix and incubate at room temperature for 5m i n . The proliferating, packaging-defective H S V -I mucoid D N A can also be used as H S V -I helper virus DNA (Fraefel 1997). b- Add 16 fxl of Lipofectamine to the other tube. mix well and incubate at room temperature for 5 m i n . 3- Mix the two tubes. Mix well and incubate for 30 m at room temperature. 4 . Add 0.9 m l of O p ti-M E M I to the tube containing the D N A - L i p o f e c t a m i n e transfection mixture. 9 ml of O p t i - M e m i . 5. Flush the V E R 0 2 - 2 cell culture (see step i) once with 2 m l of O pti-M EM I . The cells should be in a confluent state at the time of transfection. 6 - Aspirate the cell culture and add the transfection mixture. Incubate in an incubator at 37°C , 5% c 〇2 . 7 . Aspirate the transfection mixture and wash the cells 3 times with 2 m l of O p t i - M E M I . Harvesting of packaged vectors 9- Scrape off the cells with a rubber cell scraper. 10.Transfer the suspension to a 15m l conical tube. Place the conical tube in an ice bath with ice water. 11. Place the probe of the ultrasonic breaker about 0.5 cm below the liquid level of the cell suspension. Use 20% of the output power (55W). Ultrasonic breakage 20million 12.4°C, M O O g centrifugation for l0 m in to remove cellular debris. 1 3 . The supernatant obtained in the previous step was filtered into a new 1 5 m l conical tube using a ○.45 um syringe needle filter attached to a 2 0 m l -disposable syringe. 14. Remove one sample for titer determination (step 19) and divide the rest into several portions of im l each. 15. Dispense the sample into dry ice/ethanol. Store at 180 °C. Samples can also be concentrated before freezing (see below). The vector can be stored at 80 °C for at least 6 months without affecting the titer. 16. Concentrate the sample as follows. a. Transfer the carrier solution obtained in step 14 to a 3 0 m l centrifuge tube containing 2 5 % sucrose in H B S S . b- Centrifuge for 2 h with a S ○ rvaIlSS>34 turntable at 16°C, 20 000r/min. c. Resuspend the resulting precipitate in approximately 300 ul of H B S S . d - Remove a portion of the sample for titer determination (step 19) and divide the remainder into portions of 30 fXl each. e. Freeze in dry ice/ethanol and store at 80°C. Titration of Amplicon Storage Solution To detect the concentration of infectious plasmids in the amplicon storage solution, cells were deinfected with these amplicons. After 24 to 48 h , cells expressing the transgene were counted to calculate the titer, expressed as transduction units contained in each milliliter. 17. Inoculate VER 0 7 6 cells at a concentration of I X l O 5 cells per well into a 24-well plate with 0.5 ml of DMEM medium containing 10 % FBS per well. Incubate overnight at 37°C in an incubator with 5 % C O 2 . 1 8 . Aspirate the culture solution and wash each well with P B S . 19. Dilute 0 -lul, 1ul, 5ul of plasmid storage solution into 250ul of D M E M medium containing 2 % F B S, respectively, and add to the cells. 20. Incubate the cells at 37°C in an incubator with 5 % CO2 for 1~2d. 21. Detect the expression of the transgene by appropriate methods (e.g. green fluorescent protein, immunocytochemical staining, X-gal staining). 22. Count the positive cells and multiply the number of transgene-positive cells by the number of dilutions to calculate the titer of the vector (T U/mL). In human cells infected with the HSV/AAV heterozygous vector, identify the site of specificity between the vector sequence and the AAV sequence. For more product details, please visit Aladdin Scientific website.
8- Add 3.5m l of DMEM medium containing 6 % FBS. Incubate at 37°C 5% CO2 in an incubator for 2~3d.