Construction of a novel conjugative plasmid harboring a GFP reporter gene and its introduction into animal cells by transfection and trans-kingdom conjugation

We have established a novel method of gene introduction into eukaryotic cells by trans-kingdom conjugation between Escherichia coli/Agrobacterium tumefaciens bacteria and yeasts. To expand this to animal cells, we have constructed a novel conjugative plasmid, pBASGreen which contains SV40-ori/promoter, pUC-ori, IncQ type oriT/mob, Apr and Neor genes with GFP (green fluorescent protein) gene as a reporter. The introduction into COS1 and NIH3T3 animal cultured cells by conventional transfection was easily detected by fluorescence of the GFP gene product under a fluorescent microscope and the transfectants were effectively selected by the Neor marker. By the action of oriT/mob in the presence of tra genes on a helper plasmid, pBASGreen was directly mobilizable from E. coli into animal cells by trans-kingdom conjugation.