Figure 3.
Genomic characterization of the GNR23 allele, showing integration of ∼14 kb of transgene-specific DNA into the antisense strand of chromosome 5 and concomitant deletion of 25.7 kb of wild-type DNA. The 7 kb GnRH-NR transgene (A) and the integration site on chromosome 5 ∼67 kb proximal to Epha5 (B). The 5′-3′ orientation of the transgene array and the Epha5 open reading frames are indicated by arrows. Positions of flanking DNA clones isolated by Vectorette PCR (pVec-BglII, pVec-EcoRI) and by library screening (p13.1, p11.1, p10.1, p2.1, p1.2), and the markers D5Bne1 and D5Bne2 are shown relative to the transgene. Fragments of DNA are not drawn to scale. C, Markers D5Bne1 and D5Bne2, which flank the site of transgene integration, were mapped to chromosome 5 on two separate interspecific backcross panels, TJL BSS and TJL BSB. A combined BSS/BSB map is shown, centromere to top, with loci linked to D5Bne1 and D5Bne2. Genetic distances (cM) between markers are shown ± SEM. Epha5 was not mapped in either backcross panel. The Ensembl chromosome 5 physical map shows these genetic markers ordered according to their positions in the Ensembl mouse genome database (version 16.30.01). The base pair (bp) number indicates the location of the 5′ end of each marker. This illustrates the relative position of Epha5 with respect to the backcross mapping data. D, The 25.7 kb deleted region predicted by in silico analysis using the Ensembld at a base was characterized using PCR assays A-I. The positions of A, B, H, and I are depicted in B. Amplification from GNR23 DNA is successful from PCR assays that flank the deletion, A, B, H, and I, but is unsuccessful from assays C-G internal to the deletion. E, The deleted region was further characterized by Southern blot using probes located adjacent to (probes 1 and 4, B) and within (probes 2 and 3, D) the 5′ and 3′ limit of the deletion. Fragments of the predicted size were observed in both wild-type and GNR23 using probes 1 and 4. Probes 2 and 3 detected predicted fragments from wild type, but failed to detect any fragments from GNR23. F, Southern blot showing approximate size of the transgene insertion. Sites for AvrII are not contained with in the transgene, but a represent immediately adjacent to the transgene array (B), and located 16.8 kb from the distalend of the deletion (D). Probe 5 (shown in B) identifies fragments of ∼17 kb for wild-type C57BL/6J and 14 kb for GNR23. WT, Wild type.