Nuclear extracts from baculovirus-infected insect cells of the meadow moth (Sf9 ) encode the major capsid protein (V P l ) of simian virus 40 (S V 4 0 ). In the presence of M g C l 2 , CaCl2 and A T P , these nuclear extracts were able to package the superhelical structure of the plasmid D N A or R N A scrambled (R N A i ) sequences, resulting in the formation of S V 40 pseudovirosomes in vitro. This packaging method has many advantages over other viral or nonviral delivery systems. In particular, it has a wide host range and a high conversion rate. The only disadvantage of this system is the low expression per transformed cell observed in vitro , probably due to the fact that the produced D N A is confined to the cytoplasm and cannot enter the nucleus. Author: T. Friedman et al, Translated by W. Jing et al. This experiment is from "Gene Transfer".
Operation method
S V 4 0 In Vitro Delivery System Move S V 4 0 In Vitro Delivery System Materials reagents A T P (10 0 m m o l/ L; R oche) Buffer I 10 m m o l/L H E P E S (p H 7. 9) 10 m m o l/L K C l 0.lm m o l/L E D T A 0 . 1 m m ol/L E G T A Sterilize by filtration with a 0.22um membrane. Buffer I can be stored at 4°C for 30d. Add before use: l m m o l / L dithiothreitol (D T T ) Dissolve 1 tablet of protease inhibitor (Roche 1873580 ) in Iml PBS and dilute with buffer I 1 :50. Buffer II 20m m o l / L H E P E S ( p H 7. 9) 0.4 m m o l / L NaCl 0.l m m o l / L E D T A l m m o l / L E G T A Sterilize by filtration with a 0.22um membrane. Buffer II can be stored at 4°C for 30 days. Add before use: l m m o l / L P M S F l m m o l / L D T T Dissolve 1 tablet of protease inhibitor (Roche 1873580 ) in Iml PBS and dilute with buffer 11:50 CaCl2 (10 mmol/L) Cells Cell lines used for research Cell lines to be transformed are maintained in Petri dishes or suspension. Grassland nightshade moth (Sf9 ) cells Medium Cell growth medium (G r a c e insect culture with whey protein and yeastolate) Complete generation medium Use the appropriate medium for the cell line under study. M g C l 2 (200m m o l /L ) Nonidet P-40 (N P-40; 1 0% V/V in P B S ) Phosphate buffer (P B S ) Plasmid D N A (up to 17. 7k b) To obtain the highest in vitro loading efficiency, plasmid D N A was purified by equilibrium centrifugation using a density gradient of C s C l - cesium chloride. Small Interference RNA (s i RNA ) V P l Rodentivirus VPl was cloned by eliciting a DNA fragment (1463^~2770 on SV40) into the pVL1393 plasmid (PharMingen, San Diego, California), which is derived from the Aw ton ra Can a cW/omicanuclear pholyhedrosis virus (AcMNPV) (Luckow and Summers 1988). PharMingen, San Diego, California) for VPl cloning (PharMingen, San Diego, California). Instrumentation Incubator (constant humidity, 37°C, 5 % CO2) Centrifuge tubes (40 ml, sterile, Sorvall) Microcentrifuge tubes (1.5 ml) Shaker Stirred culture bottle (250m l ) Tissue Culture Dish (60m m ) Vortex mixer with microcentrifuge tube platform Constant temperature water bath (37°C) Methods Transformed Sf9 cells 1. Freshly cultured 5 X 108 Sf9 cells were infected with V P l baculovirus at an infection multiplicity of 1 0 in 250 m l culture flasks. Scaling up to 500 ml is not recommended. Nucleoprotein extraction Sf9 cells were collected 4~5d after infection. The time of cell harvesting is determined by the expression level of vpl and the packaging function of the nuclear extract. 2- Transfer the transformed cells from 250m l culture flasks to pre-cooled 40m l test tubes. 3-Prepare Buffers I and II. place on ice. Pre-cool microcentrifuge tubes. 4. Wash cells three times with cold PBS, centrifuging at 6000 rpm at 4°C for 5 millimeters each time. 5 - Remove supernatant. Vortex to completely resuspend the precipitate Do not freeze this step. 6 - Add I m l of Buffer I. Gently blow to resuspend the precipitate until it is completely dispersed. 7- Slowly add 28 m l of Buffer I. Incubate on ice for 15 m i n . 8- Add 2. 3m l of 10% NP-40. vortex for 10s. 9-Centrifuge at 13,000r/m i n a refrigerated centrifuge (~4°C) for 3m i n . 10- Remove the supernatant with a pipette tip attached to a vacuum aspirator. The supernatant is removed with a pipette attached to a vacuum aspirator. 11 - Vortex the precipitate. Add 5 m l of Buffer II. Mix thoroughly and transfer to a microcentrifuge tube. The total volume of each tube should exceed 1 ml. I2. Place the tubes on the platform attached to the oscillator. Vortex at 4°C to mix for 15m i n . 13.4 C, 13 000r/m i n Centrifuge microcentrifuge tube for 5m i n . 14- Dispense the nuclear extract supernatant into pre-cooled tubes. 15- Determine the protein concentration of the nuclear extract. Notice that the nuclear extract contains N P -4 ○. Not all methods for determining protein concentration are compatible with N 1m o . It can only be used for in vitro packaging if the concentration is greater than 3m g/m l. 16- Store nuclear extracts at 20 to 8 °C. For more product details, please visit Aladdin Scientific website.
0.5 m m o l / L phenylmethylsulfonyl fluoride (P M S F )
California). It was then digested by restriction endonucleases SmaI and BgZII (Sandalon and Opperiheim 1997, 2001). Virus reservoirs were dispensed and stored at 80°C.
