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.
Yoshida
K
K
Department of Biological Science, Hiroshima University, Japan.
Takegami
T
T
Katoh
A
A
Nishikawa
M
M
Nishida
T
T
D016475:3T3 Cells; D016960:Agrobacterium tumefaciens / Q000235:genetics; D000818:Animals; D019556:COS Cells; D003227:Conjugation, Genetic; D004926:Escherichia coli / Q000235:genetics; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection / Q000379:methods*; D015003:Yeasts / Q000235:genetics
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins