Figure 1. Generation and validation of PICH^−/− cells.

(a) Conservation of the chicken and human PICH proteins. The defined domains, designated TPR, SNF2, HELICc and PFD, are abbreviations for Tetratricopeptide repeat, sucrose non-fermenting, helicase superfamily c-terminal domain and PICH family domain, respectively. Conservation is defined as the % of amino-acid positions that are identical or from the same functional group, and is depicted as a series of peaks aligned along the PICH sequence. Data were extracted from the NCBI database. (b) The PICH gene targeting strategy at the chicken PICH locus. The black boxes represent the PICH exons and the PICH homology regions flanking the Bsr or Puro resistance genes in the targeting vectors. Positions of the 5′ and 3′ validation Southern blotting probes are shown in pink. The size and position of probed DNA that would be expected in an unmodified or a targeted PICH locus after digestion with SpeI (blue) or BamHI (orange) are shown. (c) Southern blots probing the 5′ end and 3′ end of PICH to confirm insertion of the Puro and Bsr genes in genomic DNA digested with either Spe1 or BamH1. (d) Positions of positive (1–2) and negative (3–4) control primers at the Gd PICH locus. (e) The gene knockout was validated by PCR with primer sets 1–4 on isolated genomic DNA from cells of the indicated genotypes.