FIG. 1.
Targeted replacement of Oct-6 by Brn-1 in mice. (A) Schematic representation, from top to bottom, of the targeting construct, the Oct-6 wild-type locus, and the mutant locus before and after Cre recombination. The Oct-6 and the Brn-1 open reading frames (ORF) are shown as boxes, and 10.9- and 4.6-kb-long flanking regions are shown as bars. Regions of homology between the wild-type locus and targeting vector are depicted as black bars, and surrounding genomic regions not contained in the targeting construct are depicted as open bars. Plasmid backbone sequences of the targeting construct are indicated by a thin line. Restriction sites for SacI and BglII are shown as well as the localization of the 3′ probe and the start codon of the Oct-6 gene (ATG). NEO, Neo cassette; hGH-pA, polyadenylation signal from the human growth hormone gene; loxP, recognition sites for Cre recombinase; Tk, herpes simplex virus thymidine kinase gene cassette. (B) Southern blot analysis of DNA from wild-type (+/+) and heterozygous (+/brn-1) ES cells digested with SacI and BglII for use of the 3′ probe. The size of bands corresponding to the wild type (6.2 kb) and the targeted allele (8.9 kb) are indicated. (C) Southern blot analysis of DNA from wild-type (+/+), Oct-6+/Brn-1 (+/brn-1), and Oct-6Brn-1/Brn-1 (brn-1/brn-1) mice digested with SacI and BglII for use of the 3′ probe after Cre-mediated removal of the Neo cassette. The sizes of bands corresponding to the wild type (6.2 kb) and the targeted allele (7.7 kb) are indicated. (D) Western blot of sciatic nerve extracts from 8-day-old wild-type (+/+) and Oct-6Brn-1/Brn-1 (brn-1/brn-1) mice with antibodies directed against Brn-1, Oct-6, and β-tubulin. Molecular mass markers are indicated on the left.