If I have a recombinant vaccinia virus with a gene inserted into TK locus for selection then how do I replace this gene with another gene. I came across a method called direct ligation, but I feel if this will inactivate my gene by frameshift. How do I find how many multiple cloning sites there are in between promoter and transcription start sites. Can anyone please send a reference for an exact method?
Aternatively to the genome ligation, you could clone your gene into the MCS of a transient selection (e.g. gpt selection) shuttle plasmid targeting the TK locus (with the same left/right region as the one used for the first transgene). Then perform the classical transfection/infection/clone selection (first with pressure followed by cycles without pressure) . The key is to get the transient-selection-TK-shuttle plasmid (or to make it).
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