Fig. 1.

Mapping recombinant cell lines incorporating the rat Igf1 locus. A: schematic of rat Igf1 bacterial artificial chromosome (BAC) Ch230-113M6, which consists of 241 kb of rat chromosome 7, including the ∼80 kb 6-exon Igf1 gene, ∼110 kb of 5′-flanking, and ∼51 kb of 3′-flanking DNA. The location of the pGK-Neo selection cassette, which was introduced into the BAC by recombineering (28, 62), is indicated, as are the 7 previously identified growth hormone (GH)-regulated Stat5b-binding elements (red circles) and a scale bar. The enlargement below the main map depicts the two Igf1 promoters, P1 and P2, and adjacent exons 1–3. The horizontal arrow indicates the direction of gene transcription. The locations of the 11 PCR primer pairs used to characterize each clonal cell line are indicated (see Table 1 for DNA sequences). B: results of PCR mapping experiments using genomic DNA isolated from BAC cell lines 11 and 20 and primer pairs 1 and 11. These pairs amplify DNA from the 5′- and 3′-ends of the BAC, respectively. C: results of PCR mapping experiments with genomic DNA from each BAC cell line and primer pairs 2 through 10. Collectively, results in B and C demonstrate that the entire BAC has been incorporated into each recombinant cell line. D: results of copy number determinations for cell lines 11 and 20, using as a standard curve serial dilutions of BAC DNA (see materials and methods for details).