Construction of recombinant vaccinia viruses using PUV-inactivated virus as a helper

Recombinant vaccinia viruses, (VVs) are widely used as expression vectors i n molecular biology and immunology and are now under evaluation for gene th erapy. The current techniques for inserting foreign DNA into the large VV g enome are based on either homologous recombination between transfer plasmid s and VV genomes or direct DNA ligation and packaging using replication-def icient poxviruses. Here, we describe efficient new versions of both methods that produce 90%-100% of the recombinant viruses. In the new homologous re combination method, VV DNA "arms" obtained by NotI digestion and intact tra nsfer plasmids, were used for co-transfection. ln the direct DNA ligation m ethod,foreign DNA was inserted into a unique NotI restriction site of the V V genome. In both methods, the generation of recombinant viruses was carrie d out in cells, infected with a non-replicating, psoralen-UV (PUV)-inactiva ted helper VV. The convenience, of these new techniques is demonstrated by the construction of recombinant Ws that produce E.coli beta -galactosidase. An important feature of these strategies is that any VV strain can be used as a helper virus after PUV inactivation.