A cotransfection method has been developed for the generation of recombinant baculoviruses. The permissive coleopteran cell-line DSIR-HA-1179 was transfected with a mixture of baculovirus DNA (strain PV505) and a transfer vector. The transfer vector, a pUC8-based plasmid, contained the polyhedrin gene from the nuclear polyhedrosis virus, flanked by baculovirus DNA from the dIII fragment N. baculovirus does not form plaques with DSIR-HA-1179 cells. Therefore an endpoint dilution method was used to screen for, recover and purify recombinants. There was no phenotypic character that could be used for detecting recombinants, so a dot blot assay was used to screen infected cultures for the presence of recombinants. A recombinant generated by this method contained the entire polyhedrin gene inserted at 98.03 map units from the designated start of the baculovirus physical map. No evidence of transcription or translation of the polyhedrin gene was obtained.


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