I performed a transfection to obtain a recombinant virus that expresses my protein of interest, which is marked with a yellow fluorescent protein (YFP). The PCR results showed both the band of the expected size for the incorporated fragment and a smaller band that seems to be the parental virus with the YFP but without my protein of interest (because the ban's size is comparable to the lenght of the YFP's sequence). In this case I would have a mixture of 3 viruses: Parental virus, Parental virus+YFP and the virus with the YFP and my protein of interest. The last two both present the same fluorescence at the microscope so it's impossible to tell which one I'm selecting by clonal picking.
How can I separate the virus with the protein of interest from the one that only has the YFP?
a classical method to clone a virus is also serial plaque purification (in general 3 times...). But could the "non recombinant" viruses be the result of the recombinant virus deleting the YFP insert upon passages? in that case even after cloning you will generate a population of recombinant and non-recombinant virus...
Robert Adolf Brinzer Thank you for answering. I've tried infecting a 96-well cell culture plate with different dilutions and collect the wells where there's only one fluorescent clone. Nevertheless, the mixture in the PCR results persists. I still don't understand how a single clone can present both, the band corresponding to the protein+YFP and the one correspondig only to YFP.
Didier Poncet Thanks for your answer. At first I thought the recombinant virus could be deleting the protein of interest and keeping only the fluorescent marker. However, when I collected the transfection product and performed a PCR the mixture could be evidanciated since the very beggining, even before starting the selection process. So my theory is that the site-specific recombination generated both, the protein+YFP and the one correspondig only to YFP. I would like to know your thougths on this scenario.
could the plasmid you used to do the recombination be a mixture of wt and recombinant ? (maybe the deletion of Yfp and your GOI happened during the propagation of this plasmid?) did you look whether you have a repeated sequence on each side of the insert (and also in between the yfp and the GOI) DNA polymerase could jump from one repeat to the other during replication.... maybe you could sequence the PCR fragments you got to understand what happened...
- Badges
- Science topic