Fig. 2

TK-fusion counter-selectable cassettes. a Schematic representation of counter-selectable cassettes HyTKAX, TKanAX and TKnatAX. Region used as a PCR template is shown. The backbone vector is pFA6a and the cassettes were inserted between PacI PmeI sites. Top primers anneal to the left end (pink arrowhead on the left) and Bot primer anneals to the right end to amplify indicated cassettes (pink arrowheads on the right). Cyan arrows represent diagnostic primers used for screening of correct targeting, and sequence of primers is shown in Table 2. The TK fusion genes are transcribed by the S. pombe adh1 promoter and terminated at the tef terminator. The size of the cassette is shown on the left. Transcription direction is toward right. b, c and d FdU sensitivity of cells carrying the HSV-TK fusion cassettes. Indicated cells were cultured in YES rich media, normalised and sequentially diluted five times. Five microliter of diluted fractions were spotted on YES containing the indicated drugs and incubated at 32 °C for 3 days. b Cells carrying the TKnatAX cassette are hyper sensitive to 20 μl/ml FdU. c Cells carrying the TKanAX cassette are sensitive to 100 μl/ml FdU. d Cells carrying the HyTKAX cassette are sensitive to 20–100 μl/ml FdU