Human bacterial artificial chromosomes can be targeted at Hprt by homologous recombination and, if desired, conditionally removed using cre recombinase. (a) Integration into the mouse genome of the bacterial artificial chromosome (BAC)-lacZ-reporter constructs by homologous recombination results in the human gene in either direction relative to the X chromosome; this schematic presents one possible orientation. Regardless of orientation, each insertion resulted in the presence of four loxP sites in the genome (two wild-type and one 511 mutant at one end and one wild-type at the other end of the BAC insert). (b) Crossing the BAC-lacZ-reporter females to ACTB-cre males should result in the generation of two different male offspring; BAC-lacZ-reporter animals, wild-type for the ACTB-cre transgene; and BAC-lacZ-reporter animals carrying the ACTB-cre transgene. Only the reporter animals that are positive for the ACTB-cre gene should recombine the outer most loxP sites, resulting in excision of the BAC construct from the genome and leaving one loxP site. This would result in an absence of lacZ-positive signal. hP, human HPRT promoter; h1, human first exon; m2 and m3, mouse second and third exons; mouse homology arms (dark blue); Hprt coding regions (red); vector backbone (yellow with black edges); SacB gene from BAC vector backbone (brown); 5′ and 3′ untranslated regions of the human gene (orange); coding region of the human gene (green); lacZ reporter gene (light blue). Schematic, not to scale. (c-f)lacZ expression results from AMOTL1-lacZ, MAOA-lacZ, NOV-lacZ, and NR2F2-lacZ females bred to the ACTB-cre males are presented. lacZ-positive staining (blue) was detected in AMOTL1-lacZ, MAOA-lacZ, NOV-lacZ, and NR2F2-lacZ males not carrying the ACTB-cre allele whereas absence of staining was detected in males positive for ACTB-cre by genotyping (AMOTL1-lacZ, ACTB-cre; MAOA-lacZ, ACTB-cre; NOV-lacZ, ACTB-cre; NR2F2-lacZ, ACTB-cre), suggesting whole BAC excision from the genome. Scale bar: (c-f) 1 mm. N = 3 animals for all genotypes.
Schmouth et al. BMC Biology 2013 11:106 doi:10.1186/1741-7007-11-106