Stable virus-producing cell lines for AAV assembly
Stable virus-producing cell lines for AAV assembly
Cloning of virus-producing cells by infection with a helper virus, stable virus-producing cell lines containing and genes, and r A A V (recombinant adeno-associated virus) vectors provide a reliable and efficient procedure for the establishment of r A A V repositories. However, the establishment of this class of cell lines is time-consuming. Therefore, this method is recommended only when large quantities of rAAV are required, such as in preclinical studies on large numbers of animal bodies. Author: T. Friedman et al, Translated by W. Qin et al. This experiment is from "Gene Transfer".
Operation method
Stabilized rAAV manufacturing cell construction techniques Move Stabilized rAAV manufacturing cell construction techniques Materials reagents AAV Plasmid pAAV: recombinant plasmid with rAAV vector cloned in the backbone of the plasmid pRC: plasmid containing the AAV and c a p genes expressed under its original manipulator but without the viral reverse terminal repeat sequence The nature of these plasmids will vary with the serotype of AAV created and the rAAV vector used. DMEM (Sigma-Aldrich) medium containing 10 % fetal bovine serum (research grade), 1 % penicillin (500 U/ml) and streptomycin (Cambrex, 50um/ml). G 418 storage solution (100m g/m l, m/V) Dissolve 5 g of G 418 (Invitrogen) in 50 ml of sterile distilled water, then filter the stock solution through a 0.2 Mm filter and store at 20°C. H e L a (A T C C C L - 2 ) or A 549 (A T C C C L - 185 ) Cells Thiamphenicol B storage solution (l O m g /m l , m/V ) I g of chlortetracycline B (Invitrogen) was dissolved in IOO ml of sterile distilled water. The stock solution was then filtered through a 0.2 um filter and stored at 120°C. The solution was then stored in a sterile vapour chamber at 0.2 ml of sterile distilled water. Selective plasmids pPGK-neo: This plasmid encodes neomycin resistance under the control of the murine phosphoglycerate kinase-1 promoter. p S V - h y g r o : This plasmid encodes a thaumatin resistance gene under the control of the viral S V 4 0 promoter Any recombinant plasmid that expresses a selective marker can replace these two plasmids. I X Trypsin / E D T A Solution Dilute 10X Trypsin / E D T A Storage Solution (Sigma-Aldrich) with sterile 0.9 % NaCl and store the solution at -20 °C. Wild-type adenovirus 5 (A d 5) (A T C C V R -5) METHODS Construction of stable packaging cell lines 1 . 24 h prior to transfection, inoculate HeLa or A 549 cells in IOc m tissue culture trays at a density of 3 XIO6 cells. The density of each cell line is different and it is important to ensure that the cell confluence is 50 % to 80 % before transfection. 2. Co-transfect cells with 10 ug pRC and I.0 ug pPGK-neo using CaPO4. 3. 24 h later, trypsin digested the cells, gradient diluted the suspended cells (1/5, 1/10, 1/20, 1/40), and then re-inoculated each diluted cell with IOcm cell disks (two copies). Incubate the cells for 20 to 24 h. 4 . Aspirate the cell culture medium and add fresh medium containing l m g / m l G 418. 5. Continue to culture the cells for 3 to 4 weeks, replacing the medium containing l mg/ml G 418 every 2d. A large number of cells die within 7 to 14 d after transfection, and monoclonals begin to appear 3 to 4 weeks after transfection, during which time it is important to maintain selective pressure on cells for G418. 6- Observe and label individual clones from the bottom, and then isolate the monoclones. After trypsin digestion of the clones, use a cloning loop or pipette tip to scrape the monoclones from the culture plate. 7- Expand the single clone by incubating the cells in G 418-free medium to spread the clone over two IOc m cell plates. 8-Collect the cells from one IOc m plate and freeze them at 80°C (approx. 5X 106 cells per vial) and prepare genomic DNA from the cells in the other plate. Screen for clones with existing and/or cap D N A 9- Prior to 24 h of viral infection , melt and amplify PCR-positive clones, and then inoculate the cells into two wells on a 6-well cell plate (approximately 5X 105 cells per well). The clones were screened by amplifying r-print and/or DNA using appropriate primers modeled on genomic DNA, and the PRC plasmid was used as a positive control. 10. For HeLa and A549 cells, infect one well of cells with wild-type Ad5 at an infection multiplicity of 50. The number of infected cells was adjusted so that 90% of the cells were infected and the lesions were clearly observed in the infected cell wells. For more product details, please visit Aladdin Scientific website.
Cell Culture Media
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