Ref ID : 7338
W. John Haynes, Kit-Yin Ling, Yoshiro Saimi, and Ching Kung; Induction of Antibiotic Resistance in Paramecum tetraurelia by the Bacterial Gene APH-3'-II. J.Eukaryot.Microbiol. 42(1):83-91, 1995
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We have generated a transformation marker for Paramecium using a Paramecium expression vector (pPXV) and the open reading frame (ORF) of the bacterial antibiotic resistance gene aminoglycoside 3'-phosphotransferase-II (APH-3'-II or neo(r)) from the transposon Tn5. The expression vector contained a small multiple cloning site between the 5' and 3' non-coding regions of the calmodulin gene, and Tetrahymena telomere sequences for the stability of the plasmid in Paramecium. After the neo(r) ORF was inserted, the plamid was referred to as pPXV-NEO. Delivery of approximately 10-20 picoliters of linearized PXV-NEO at >/_2000 copies/pl into the macronucleus effected 100% transformation. Southern and Northern blot hybridization showed the presence of neo(r)-specific DNA and RNA, respectively, in all of the transformed clones but not in the untransformed clones. The degree of resistance to G-418, and the concentrations of neo(r)-specific DNA and neo(r)-specific RNA in the clones were proportional to the concentration of the vector injected. We have demonstrated that when the linearized plasmid was injected into the macronucleus, the prokaryotic sequence conferred an antibiotic resistance to Paramecium despite codon-usage differences.