Construction and initial characterization of HTETOP cells. (A) Principal of RNAse protection assay use to detect endogenous (top) and exogenous (bottom) topoIIα transcripts. The 5′ ends of transcripts are shown as shaded boxes, numbered according to exons. The exogenous transcript has non-topoIIα-derived sequences at its 5′ end (unshaded box). Protected and unprotected regions of the RNA probe are shown above each transcript (solid and broken lines, respectively). (B) Products of RNase protection assay outlined in A were separated by denaturing PAGE. Protecting RNA was from the indicated cell lines grown in the presence or absence of doxycycline for 2 d. (C) Gene targeting strategy. Bold lines represent 5′ regions of TOPOIIα genes with exons 1–4 (left to right) shown as black boxes. Pairs of TOPOIIα alleles in HTETOPwt (+/+) HTETOPhet (-/+) and HTETOP (-/-) cells are shown with relevant sites for PstI (P), and the resulting fragment sizes are indicated. Linearized targeting constructs are shown with TOPOIIα DNA aligned to TOPOIIα target alleles, and the position (disrupting exon 4) and orientation (opposite to TOPOIIα) of their gpt and zeo selection cassettes are indicated; remaining plasmid DNA is shown as a thin line. The vertical dotted line indicates the limit of homology between targeting constructs and TOPOIIα alleles. The probe (Pr) used for Southern analysis (gray bar) and oligonucleotides (O1, O2, and O3) used for PCR assays (arrowheads) are shown aligned with homologous DNA. (D) Southern analysis. PstI-digested genomic DNA isolated from HTETOPwt (+/+), HTETOPhet (-/+), and HTETOP (-/-) cells, and labeled marker DNA (M), were separated, blotted, and probed with the probe shown in C. (E) HTETOPhet doubling times during continuous culturing (see Materials and Methods) with (open circles) or without (squares) added tetracycline at 1 μg/ml. (F) HTETOP doubling times, grown as in E but with tetracycline at 0 (squares), 1 (diamonds), or 2 (circles) ng/ml.