I studied the procedure of recombinant adenovirus production but I couldn't understand why a shuttle vector is being used for cloning of the insert and then the adeno-backbone vector and the shuttle vector will be co-transformed in the e.coli for a recombination process to happen and then it will be linearized for packaging in HEK cells.
why they don't insert the foreign DNA directly into the backbone plasmid and then linearize it because the recombination between the shuttle and backbone isn't such a high rate process and it is superfluous, in my opinion!
I haven't done any practical experiment for Adenovirus packaging yet! However, As I know, you need just a shuttle vector_exp. pAdTrack-CMV_ in E.coli. after recombination with the backbone vector, exp. pADEasy-1, you should linearize it and transfect it to HEK cell for packaging.
But it isn't related to my question.
One reason is the pADEasy plasmid is ~33kb in size. The ligation efficiency with such a large vector is very low and are limited by the number of useful restriction sites. However, it is much easier to clone your gene of interest into the shuttle vector (~7.5 kb) because it is much smaller and has been engineered with a nice multi-cloning site. Once you have the shuttle vector, you can use the power of the E. coli homologous recombination system to make a adenovirus genome expressing your gene of interest.
Thank you for your answer. Yes, I think one of the reasons is the size of the pAdEasy and the low efficiency of ligation. However, the efficincy of homologous recombination is also very low. Moreover, If it was supposed to use just ADEasy vector, it could be modified and optimized with a better mulitple cloning site. I don't think this is the only reason that this system is being used and no one wants to optimize it.
I am working with this tool: shuttle vector and p-aeasy-1 to produce adenovirus + mutated GFP (insert).
Once we have the virus produced in HEK, my question is if we can infect using this virus stock other cell types such as MRC-5 or HeLa to carry out other experiments.
Thank you very much,
Cheers.
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