Generation of Survivin conditional knockout in DT40 cells. (A) Diagram of the survivin locus and targeting vectors used. Arrowheads, EcoRI cleavage sites. (B) Strategy for rescue and shutoff of Survivin expression. (C) Table showing constructs used for the two knockout cell lines. (D) Southern blot of wild-type, heterozygote, and Survivin-null clones. EcoRI-digested genomic DNA was hybridized with the 5′ external probe (red bar) shown in A. Superscripts 1 and 2 refer to heterozygotes from knockouts 1 and 2, respectively. (E) Repression of rescue Survivin mRNA confirmed by RT-PCR of total RNA from heterozygote and KO1 cells incubated with doxycycline. (F) Real-time PCR confirms survivin repression by doxycycline. Values were normalized relative to actin mRNA. Values for cells grown in doxycycline are shown as striped bars. Error bars indicate SD. (G) Immunoblotting analysis of Survivin repression for KO1. 20 μg of whole cell lysate from DT40 (wild type [WT]) and Survivin^OFF cells was subjected to 12.5% SDS-PAGE and probed with affinity-purified polyclonal anti-Survivin antibody. Loading control, anti–α-tubulin. (H) Immunoblotting analysis of Survivin repression for KO2 performed as for G.