Polyomavirus: SV40
Polyomavirus: SV40
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 of SV40-derived vectors Move Construction of SV40-derived vectors Materials reagents Cells were cultured in 75 cm2 cell culture flasks with DMEM-IO and passaged every 3~4d. 30 g and 40 g of solid CsCl were dissolved in 70 ml and 50 ml of 0- 02 mol/L Tris-HCl (pH 7. 4), respectively. Im l of each solution was taken and weighed on an analytical balance to determine density. The solutions were sterilized by filtration using a 0.22Pl membrane. Dual Decontaminants 5 % Decholate (Sigma-Aldrich) 10% Triton X-IOO (Sigma-Aldrich) Dulbecco's basic medium (D M E M ; G I B C O B R L ) Sterilize by filtration with a 0.22um membrane. Ethanol (70% V/V) F u G E N E -6 Transfection Reagent (Roche) Growth medium (p H 7. 4) (D M E M -10) D M E M 10 % normal calf serum (C a m b r e x ) 1 % Penicillin/Streptomycin (Cellgro) 1 % L-glutamic acid (Cellgr0) Sterilize by filtration with a 0.22um membrane. Maintenance medium (p H 7. 4) (D M E M -2) D M E M 2 % normal calf serum (C a m b r e x ) 1 % Penicillin/Streptomycin (Cellgro) 1 % L-glutamic acid (Cellgr0) Sterilize by filtration with a 0.22pm membrane. 10 X phosphate buffer (P B S ) 2 g K C l 80 g NaCl 14.4 g N a 2 H P 0 4 2.4 g K H 2P 0 4 Dissolve in IL double distilled water (WdH2O). Prior to use, dilute 10 X reservoir solution with double-distilled water to I X PBS. adjust pH to 7.4. sterilize by filtration with a 0.22 pm membrane. rSV40 DNA Viral genomic DNA containing the desired transgene is excised from the vector plasmid, gel purified, and then recirculated using standard techniques. Trypsin/ E D T A (Cellgro) Water, double-distilled Instrumentation Cell scraper (sterilized, separately packaged) Centrifuge tubes (plastic, clean, and clear; b e c k m a n ) Sterilize centrifuge tubes by soaking in 70% ethanol for at least 15 minutes at room temperature before use. CO2 incubator (maintain 37°C, 5 % to 6 % C02; NAPCO) Freezing tube (I.5mL; Nalgene) Dialysis buoys and dialysis cards (0.5-3.O m l ; Pierce) 28. Use the following formula to calculate the number of virus particles per milliliter: 29. The purity of the sample is verified from the dissociation curve. A single peak indicates a PCR product. For more product details, please visit Aladdin Scientific website.
COS-7 Cells (A T C C )
Cesium chloride gradient solution (1.28 g/m l and 1.50 g/m l)
Initial number of copies X 5X 500 = Number of viral copies Anl
The purity and size of the PCR product can also be determined by agarose gel electrophoresis.