Ptet off

AML12-derived stable cell line AML12HBV_DE11, AML12HBVpolR105A and AML12HBVcore were established by co-transfection of AML12 cells with plasmid pTRE_HBV_DES, pTRE_HBV_DEpolR105A or pTRE_HBVcore with pTet-off (Clontech) at a molar ratio of 10:1. The transfected cells were selected with 400 μg/ml G418 in the presence of 1μg/ml tetracycline. G418-resistant colonies were picked and expanded into cell lines.

The plasmid carrying 1.6 kilobase pairs (kb) TYMS cDNA fragment, pcHTS1, has previously reported [10 (link)]. From this plasmid, a 1.0 kb fragment was amplified by polymerase chain reaction (PCR), using mismatch primers that alter the original Kozak-like motif in the 5’ untranslated region to the Kozak consensus sequence [11 (link)] and generate new restriction sites, NheI and EcoRV (Fig 1A). This NheI-EcoRV fragment including the modified TYMS cDNA, TSCD3, was subcloned into the cDNA expression vector in the Tet system, pTRE2hyg, which is commercially provided by Clontech Laboratories Inc. (Fig 1B). The constructed vector was designated as pTRE2hyg-TS3. The vectors expressing the Tet transactivators, pTet-ON and pTet-OFF, were also obtained from Clontech Laboratories Inc.