Figure 1

Generation of a DBP-minus mouse strain. (A) Strategy for the replacement of the DBP gene by homologous recombination. A DBP targeting vector, containing a bacterial lacZ and a neo cassette between DBP upstream sequences (encompassing 6.5 kb of 5'-flanking sequences and 120 bp of 5'-non-translated sequences) and DBP downstream sequences (containing the last three DBP codons, 900 bp of 3'-non-translated sequences and 300 bp of the 3'-flanking region), was electroporated into ES cells. Homologous recombination by a double crossover event lead to the replacement of the entire DBP coding sequence with the lacZ–neo sequences. The positions of some restriction sites are depicted. The following abbreviations are used for restriction endonucleases: E (EcoRV), B (BamH1), Bs (BstEII), N (NotI) and P (PvuI). Note that the NotI sites are vector-borne (boundaries of cloned DBP gene region) and do not exist in the genome. LACZ stands for the bacterial -galactosidase gene; HSVI–TK stands for the thymidine kinase gene of herpes virus I. NEO stands for the neomycin resistance gene, and UMS is a sequence that was inserted to terminate upstream transcripts (but in our hands this sequence was ineffective). The positions of PCR primers and DNA probes used in the identification of recombined alleles are indicated. (B) Southern blot analysis of DNA from F₂ mice. Heterozygous DBP animals were obtained from matings between male mouse chimeras producing 129/Ola DBP mutant gametes with 129/Ola females producing wild-type gametes. The DNA of F₂ animals obtained from isogenic F₁ 129/Ola males and females, heterozygous for the DBP mutant allele, was analyzed by Southern blot analysis of DNA digested with the restriction endonuclease BamHI. An example for each of a wild-type mouse (DBP+/+), a heterozygous mouse (DBP+/-) and a homozygous mutant mouse (DBP-/-) is shown. The position of the genomic hybridization probe is indicated in (A).