Generation and characterization of an INCENP promoter hijack–conditional knockout cell line. (A) Map of the chicken INCENP locus showing the region replaced in the promoter hijack, the size of the resulting restriction fragments, and the probe used in Southern analysis (red box). (B) Southern analysis showing the first targeting (PuroR) and the digestion after removal of the puromycin marker (PuroS; Samejima et al., 2008). (C) Map of the chicken INCENP locus showing the region targeted by the gene disruption construct. (D) Southern analysis demonstrating targeting of the gene disruption construct. Only one band is observed because the probe (red bar) recognizes a region deleted in the promoter hijack of the first allele. KO, knockout. (E) Statistical summary of the targeting efficiencies. (F) Relative mRNA level of incenp and incenp-like gene expressed in wild-type clone 18 cells. Three independent RNA preparations were used in three independent experiments. (G) Growth curves of DT40 wild-type (WT; clone 18), INCENP^ON, and INCENP^OFF cells. Wild-type and INCENP^ON cells have a similar proliferation rate. INCENP^OFF cells cease proliferation 12 h after doxycycline addition. (H) Immunoblot showing that endogenous INCENP is efficiently depleted by 20 h after the addition of doxycycline. α-Tubulin was used as a loading control. (I) INCENP^OFF cells are defective in completion of chromosome alignment. Live cell imaging of INCENP^ON/OFF cells stably expressing histone H2BRFP revealed that INCENP^OFF cells stay for a statistically longer period in prometaphase before entering to anaphase. Images were taken every 2 min. (B and D) X indicates the promoter hijack allele. Error bars indicate SD.