Targeted disruption of Cybrd1. (A) Cybrd1 wild-type locus and (B) targeted locus after introduction of an intronic floxed neomycin (Neo)/cytosine deaminase (CD) cassette and loxP sites (yellow triangles). Positions of 3′ and internal probes used for Southern blot analysis are shown as red bars A and B, respectively. (C) Cybrd1 locus after complete excision to inactivate the gene. Squiggled lines to left of diagrams in A-C indicate segments of DNA not included in the figure. Pink bars indicate the size of fragments digested by SacI using a 3′ probe by Southern analysis, and blue bars indicate the length of fragments digested by SphI using a 5′ probe by the same analysis. (D) Southern blot analysis of the 3′ end of the SphI digested locus in ES cells after Cre-mediated excision, using probe A. Clone 6 retains the floxed allele; clones 2, 4, and 5 have undergone complete excision, deleting exon 2. Clones 2 and 4 were used to obtain Cybrd1^-/- mice without residual Neo and CD cassettes. (E-F) Southern blot analysis of the 3′ end of the SacI digested locus in genomic DNA using probe A (E), and the internal probe B within exon 2 (F). Lanes 1, 3, 5, and 6 show wild-type mice, 2 and 8 show homozygous Cybrd1^-/- mice, and 4 and 7 show heterozygous Cybrd1^-/+ mice.