Preservation and amplification of targeted modified measles viruses
Preservation and amplification of targeted modified measles viruses
The S T A R System (6 Histidine Labeling and Retargeting) employs a pseudo-receptor and a label for a virus to enable the rescue and amplification of fully recombinant viruses that are no longer able to bind to the natural measles virus receptor. This protocol provides detailed instructions on how to use this system to rapidly generate newly targeted viruses in three simple steps. First, the c D N A encoding the targeting ligand is cloned into a chimeric H-protein shuttle vector capable of encoding a polypeptide tag with six histidines at the C-terminus.
Author: T. Friedman et al, Translated by Wei Qin et al. This experiment is from "Gene Transfer".
Operation method
Improved rescue and colonization of measles-trending viruses Move Improved rescue and colonization of measles-trending viruses MATERIALS Particular care should be taken when using the readable frame of the ligand (Fig. 1). For effective rescue, the genome length of measles virus produced by genetic engineering should be a multiple of six (hexamericlength). We therefore constructed two shuttle vectors that contain or do not contain a 3bp deletion in the noncoding region of the H protein mRNA, relying on ligands containing an odd or even number of amino acids, using the PTNH6-Haa (even) or pTNH6-Haa (odd) plasmids, respectively. 2. Insert a digested fragment of PAD/S BUCKLE I encoding the chimeric H protein into the p (+) M V e G F P site of the cDNA clone of the full-length leprosy virus genome. Transfection of Pendulum Rescue Virus 3. One day prior to transfection, trypsin digestion was performed on fused 293-3-46 cells collected in IOO m m dishes and resuspended in 12 m l of complete culture medium containing I.2 m g / m l G 418. 4.6 Add 2 ml of resuspended cells to each well of the plate and incubate at 37°C for 20 to 24 h. Incubate at 37°C for 10 minutes. 5- 3~6 h before transfection, aspirate the cell culture solution from the 6-well plate and replace it with 2 ml of complete culture solution without G 418. 6. Dispense 250M 12X HBS into a sterile tube. 7. In a second sterile tube, mix 5M g of recombinant full-length measles virus cDNA with 5~50 g/L of expression vector p EMC-La and add water to a total volume of 225ul. 8. Add 25ul of 2. 5m o l / L CaCl2 to the D N A solution. 9- Add D N A solution (step 8 ) to 2X H B S (step 6) dropwise while stirring. 10. Incubate the mixed solution for 30 m i n at room temperature and vortex to mix. 11-Immediately add the solution dropwise to 293-3-46 cells in a 6-well plate. Incubate at 37°C for 14-16 h. 12. Replace the transfection medium with fresh G 418-free complete culture medium. 13- Wrap the transfected 6-well plate with sealing film. 43. 5°C water bath for 3 h to induce thermal stimulation. After the water bath, the plates were incubated in a 37°C incubator until the cells were harvested. 14. V e r o-a H i s cells were collected by trypsin digestion the day before coverage (e.g., 48 h after transfection). 15-Transfer the V e r o cells to I O m m culture dishes to achieve a concentration of I.5 X 105 ~ 2 X 106 cells/well. Then add complete culture medium containing G 418. Incubate at 37°C for 14 to 16 h. Cell confluence should be approximately 70% at the time of transfection. 16. 72 h after transfection, blow the cells with a pipette to disperse the cells, and disperse the monolayer into small pieces. Place the dispersed cells in 5 ml of culture medium. Do not use cell dissociators. 17. Cover 293-3-46 cell suspension with Vero-aH i s monolayer cells cultured in I o o m m dishes. Incubate at 37°C for 2 to 7d. Several centers of infection can be observed as multinucleated syncytia, and elevated green fluorescence expression can be seen under a fluorescence microscope. Proliferation after virus rescue 18. V er ○-aH i s cells were collected by trypsin digestion the day before transfer of harvested virus spots. 19.Transfer V e r o cells to a 6-well plate to a concentration of 2 X IO5 cells/well. Then add 2 ml of complete culture medium containing G 418. Incubate at 37°C for 14-16 h. For more product details, please visit Aladdin Scientific website.
Cell confluency should be approximately 80% for transfection.
