FIG. 1.
POT1 gene-targeting strategy. (A) Organization of the chicken POT1 gene locus (black line), the gene replacement, and the gene disruption construct. Black boxes in the gene locus represent exons (E1 to E7), the initiation codon in E4 is marked, and the bar represents the intron 3 probe. The white box represents the ER-POT1 and neomycin (Neo) expression cassettes in the gene replacement construct and the Bsr resistance gene in the disruption construct. Gray boxes represent segments of POT1 genomic DNA. Regions of homology with the wild-type gene locus are marked by dashed lines. (B) Southern blot of BglII-digested genomic DNA hybridized with probe to intron 3. Lane 1, 1-kb marker; lane 2, wild-type (WT) DT40 cells; lane 3, ER-POT1 replacement cell line; lane 4, conditional cell line with POT1 disruption and ER-POT1 replacement. (C) Western blot of total nuclear proteins from WT and ER-POT1 cells probed with POT1 antibody. Nuclei (5 × 106) were loaded in each lane. The arrow marks endogenous POT1 (∼87 kDa), and the arrowhead marks ER-POT1 (∼127 kDa).