A NOVEL VACCINIA VIRUS EXPRESSION SYSTEM ALLOWING CONSTRUCTION OF RECOMBINANTS WITHOUT THE NEED FOR SELECTION MARKERS, PLASMIDS AND BACTERIAL HOSTS

Vaccinia virus is one of the most widely applied expression systems fo r use in eukaryotes in molecular biology. Expression of heterologous g enes in the vaccinia virus system, however, requires integration of th e foreign DNA into the vaccinia virus genome by means of homologous re combination or by direct molecular cloning. In both cases, plasmid vec tor constructs are required that contain the gene of interest and, usu ally, a marker gene, both of which are controlled by suitable promoter sequences. In order to simplify the construction of recombinants and to eliminate the need for a marker gene we have developed a modified v accinia virus genome that allows the direct targeted insertion of DNA fragments downstream of a strong vaccinia virus promoter without any f urther cloning steps. The gene of interest is amplified by PCR using o ligonucleotide primers that provide an SfiI site at the 5' end and an RsrII site at the 3' end of the PCR product. Following digestion with these restriction enzymes, the PCR product is operationally linked to a synthetic early/late promoter within the viral genomic DNA via the u nique SfiI/RsrII sites of the modified vaccinia virus genome. Using th is approach, intermediate plasmid constructs and bacterial hosts are n ot required and time consuming screening steps can be omitted, because 90% of the virus progeny carry the foreign DNA.