This chapter describes the construction of a fusion-mediated vector based on inactivated H V J (hemagglutinating virus of Japan, also known as Sendaivirus). The H V J liposome consists of a DNA-containing fusion liposome and inactivated H V J. The H V J packaging vector is a much simpler vector that is formed by packaging DNA directly into inactivated H V J and does not require the involvement of liposomes. Author: T. Friedman et al, Translator: Jingwei et al. This experiment is from "Gene Transfer".
Operation method
Preparation of H V J liposomes and H V J packaging vectors for gene delivery Move Preparation of H V J liposomes and H V J packaging vectors for gene delivery Materials reagents B S S (Buffered Salt Solution) 137 m m o l / L NaCl 5.4 m m o l / L K C l 10 m m o l / L Tris-HCl (p H 7. 6) Dissolve 8 g NaCl, 0.4 g KCl and I.21 g Tris-HCl in distilled water, adjust p H to 7.6 with lmol/L hydrochloric acid, and then volume with distilled water to 1 L. Autoclave and store at 4°C. Bovine brain phosphatidylserine sodium bile salt (PS), chromatography pure (Avanti Polar Lipids Inc.). Chloroform (Sigma-Aldrich) Cholesterol (Sigma-Aldrich) D C - Cholesterol (Avanti Polar Lipids Inc.) D M S O (dimethyl sulfoxide) (Sigma-AWr i c h ) D O P E (1, 2-dioleoyl phosphatidyl ethanolamine) (Sigma-Aldrich) Nitrogen Phosphate Buffer (P B S) Lecithin from chicken egg yolk (P C) (Sigma-Aldrich) Plasmid D N A Plasmid DNA is purified by column chromatography and dissolved in TE (tog/ml) buffer, BSS or water and stored at 20°C. The final concentration of DNA should be greater than lmg/ml. The final concentration of DNA should be greater than lmg/ml. Polypeptone Solution 1 % polypeptone (pancreatic digest of casein; Wako, Osaka, Japan) 0 . 2 % NaCl (p H 7.2) Dissolve 5 g of polypeptone and Ig NaCl in distilled water, adjust the pH to 7.2 with l ml/L NaOH solution, then volume to 500 ml with distilled water, autoclave and store at 4 °C. Ichthyocyanine sulfate (l0 mg/m l, dissolved in PBS) Sphingolipid (Sigma-Aldrich) Sucrose gradient solution 150 g and 300 g of sucrose were dissolved in BSS, then volume to 500 ml with BSS, autoclaved and stored at 4℃ to obtain Sucrose solutions of 30 % and 60 % (m/V) were obtained. T r i s - E D T A (T E ) 10 m m o l / L Tris-Cl (p H 8. 0) lmmol/』L E D T A TritonX-IOO (2 % in TE solution) Instrumentation Fibrin acetate membrane (○.45um and 0.200 um) (Nalgene) Centrifuge (J2-HS) with J A-20 rotor and 35 ml centrifuge tubing Low-speed centrifuge (05P R-22, Hitachi, Tokyo, Japan) Separation column (endomycin-free, used for plasmid DNA preparation) (QIAGEN) Plastic centrifuge tube (50m l) (Becton-Dickinson) Glass tubing (24mm diameter, 12cm long) (Fujiston 24/40, Iwaki Class C a l t d , T o k y o , Japan) These glass tubes can be custom made or sterilized tubes similar to those resistant to chloroform corrosion can be used. New tubes are soaked in saturated KOH-ethanol solution for 24 h before use, washed in distilled water and heated at 180° C for 2 h. The tubes are then heated at 180° C for 2 h. The tubes are then cleaned with distilled water. Spectrophotometer (DU-68, Beckman Instruments) Rotary evaporimeter (Model S R-6500, T okyo Rikakikai Inc., T okyo, Japan) Ultracentrifuge with RPS-40T rotor (55P-72, Hitachi) Ultraviolet crosslinker (Spectrolinker X L-1000, Spectronics CO . ) Vacuum Pump with Pressure Gauge (Model Asp-13, Iwaki-Glas Co., Ltd., Togyo, Japan) Ltd., Tokio, Japan) METHODS Preparation of HVJ in eggs that are about to hatch from chicks 1- The H V J virus seeds were thawed rapidly and then diluted with polypeptone solution in the ratio of 1 : 1000. The diluted seeds were stored at 4°C , until the next step of the experiment was started. 2. The chick embryo was observed with light in a black chamber at room temperature. A point for injection was marked at about 0.5m m above the chorionic allantoic membrane, and the puncture was made at the marked point after disinfecting it with tincture of iodine. 3- Inject 0. I m l of diluted virus seed into each egg using an I m l sterile syringe with a 26-gauge needle. Insert the needle vertically in order to puncture the chorionic allantoic membrane. 4 - After inoculation with virus seed, cover the hole in the egg formed by the injection with melted paraffin wax and incubate the eggs for 4d at 36.5 °C and sufficient humidity. Pre-cool the eggs at 4°C for at least 6 h before harvesting the virus. 5 . Harvesting the virus: Peel a portion of the egg shell and transfer the chorionic allantoic membrane embryo fluid into a sterilized vial using an IOM syringe with an 18-gauge needle and store at 4°C to avoid freezing. This can be done at room temperature and the virus is stable in the liquid for up to 3 months. Purification of Chorionic Allantoic Membrane Embryonic Fluid H V J 6 . 2 0 0 m l of chorionic allantoic membrane embryonic fluid was dispensed into four 50 m l conical centrifuge tubes and centrifuged for lO m in with a low-speed centrifuge at 3 0 0 0 r / m in (IO O O O gO ) at 4°C . 7- Dispense the supernatant into 6 centrifuge tubes (B e c k m a n J & 2 0 ) and centrifuge for 3 0 m in at 4 °C , 15 0 0 0 r / m in ( 2 7 OOOOg) . 8. Add 5 ml of BSS to the precipitate in each centrifuge tube and allow to stand overnight at 4 °C. 9. Gently resuspend the precipitate, divide the suspension between two centrifuge tubes and centrifuge according to the settings in step 6. 10. Add 5 ml of BSS to each tube and incubate at 4°C for more than 8 hours. 11. Gently resuspend the precipitate and centrifuge for 15 m in at 4°C, 300 r/m in (IOOO g). Transfer the supernatant to a sterile tube and store at 4°C. 12. After the supernatant has been diluted 10 times, the concentration of the virus is determined by measuring the absorbance at 540 nm with a spectrophotometer. HVJ can be inactivated by ultraviolet crosslinking at 99 mjoules/cm2 . One optical density unit at 540 nm is comparable to 15,000 erythrocyte cohesion units, and the erythrocyte cohesion units reflect the activity of the fusion. Supernatants prepared as described above typically show 20,000 to 30,000 HAU/mL. aseptically prepared viral solutions maintain fusion activity for 3 weeks, and HVJ inactivated with UV light can be stored in 10 % DMS O at either 80°C or 170°C (in liquid nitrogen) for more than 6 months. Preparation of Lipid Complexes 13- Lipid solution was prepared by dissolving D O P E (12. 2 mg), sphingosine (11.5 mg), and cholesterol (23.8 mg) in 38700 ul of chloroform; I O O mg of P C was dissolved in I m l of chloroform, and 130 ul was mixed with 3870 ul of lipid solution. The resulting 4OOOul mixture is referred to as the basic mixture of liposomes, and more advanced mixtures are used to prepare anionic or cationic liposomes (as shown below) or are stored at 20°C under nitrogen. 14 . Anionic liposome mixtures are prepared by adding IO mg of PS to the above basic mixtures; cationic liposomes are prepared by adding 6 mg of DC-cholestrol to the above basic mixtures. 15- Dispense the resulting lipid solution into 8 glass tubes, each containing 0.5 ml of 8.8 mg of the lipid substance, and store the tubes on ice or in nitrogen at 20°C until the solvents have evaporated. Lipid solutions should have the solvent evaporated as soon as possible. 16 . Connect a glass tube to a rotary evaporator, immerse the end of the tube in a water bath at 40°C, and evaporate the organic solvent under vacuum conditions for 5 to lOmin. Lipids suitable for liposomes are those that adhere to the inner wall of the glass tube and form a thin layer; lipids deposited at the bottom of the tube are not suitable. The lipids are stored under nitrogen at 20°C for 1 month. Preparation and delivery of HVJ carriers 17- Preparation of HVJ carriers for fusion-mediated gene transfer a. Plasmid DNA (200/ig of DNA in 200 M) was added to a lipid mixture in a glass tube (step 16), vortexed for 30 s, and incubated at 37°C for 30 s. This cycle was repeated eight times. In this way, plasmid DNA (up to 20 kb) can be encapsulated by anionic liposomes with an efficiency of 10 % to 30 % or by cationic liposomes with an efficiency of 50 % to 60 %. b. Liposome suspensions were passed through a 0.45um pore size acetic fibrin filter membrane and then through a membrane with a pore size of 0.2um to obtain liposomes of uniform particle size. Liposome preparation using an extruder with a polycarbonate membrane resulted in more uniform particle size. c- Add 15 OOOHVU of UV-inactivated HVJ virus to the liposome suspension, place the glass tubes on ice for 5 to 10 minutes, and incubate for 1 hour at a constant temperature of 37°C in a shaking water bathtub (speed 120/min). d. Fill the centrifuge tubes with Iml60% sucrose solution, then with 70% sucrose solution, cover with a HVJ-liposome mixture, and finally fill the tubes with BSS. e. Separate the HVJ-liposome complex from HVJ by gradient centrifugation with sucrose at 4°C on a 62-octoglycerol strip. For more product details, please visit Aladdin Scientific website.
H V J seeds from H V J (Z system, V R -105, A T C O)
The best H V J seeds were stored in 10 % D M SO in 100ul portions.
(Beckman Instruments)
Ultracentrifuge tube (12 ml) (Hitachi)
Preparation of DNA-containing HVJ liposomes (Figure 2)
