Reph, a Regulator of Eph Receptor Expression Figure 3. Isolation of eph and ephrin mutants. A) The 10.2 kb eph locus (topmost diagram) consists of 14 exons (boxes; white = UTRs, black = coding sequences) and 13 introns (lines), localized to the 102D2-D5 region of chromosome IV. The core donor construct (see Methods for details) consisted of eph genomic sequences (blue) lacking exons 1–4 (deleting the 59UTR, start codon, signal sequence and a portion of the exoplasmic domain) and a 39 region lacking the kinase domain and terminal 39 sequences. An I-SceI site was engineered into the middle of exon 6 (red shading). The core construct was placed upstream of a white gene marker (whs, green shading), the whole being bracketed by FLP recognition target sequences (FRT, purple shading) and inserted into the transformation vector. ‘Ends-in’ recombination induced by FLP and I-SceI resulted in partial tandem duplication of the eph locus (bottom-most diagram). B) Southern blot analysis of five candidate ephKD targeting events. Lane 1: molecular weight markers. Lanes 2–8: NotI digests of genomic DNA derived from: L2 (Canton S, control), L3 (Donor line, control), L4 (ephKD1), L5 (ephKD2), L6 (ephKD3), L7 (ephKD4) and L8 (ephKD5). The lower molecular weight band (non-mobilized donor construct) was absent from ephKD lines 1, 2 & 4 indicating successful homologous recombination. Lanes 9–15: BglII digests of genomic DNA derived from: L9 (Canton S, control), L10 (Donor line, control), L11 (ephKD1), L12 (ephKD2), L13 (ephKD3), L14 (ephKD4) and L15 (ephKD5). The convergence of distinct donor and endogenous eph bands into a single band due to homologous recombination is clearly evident in the ephKD1, 2 & 4 lines (L11, L12 and L14). C) RT-PCR using primers for the full- length eph transcript (L1–9, left side of gel and left diagram; primer locations indicated by arrows). L1 (Canton S, control), L2 (Donor line, control), L3 PLoS ONE | www.plosone.org 6 May 2012 | Volume 7 | Issue 5 | e37303