Fig. 1. Generation of a Cre-mediated knockout of the Bmpr gene. (A) The loxP/Cre system requires, at the minimum, two pedigrees of animals: an activator strain that directs the tissue-specific expression of the Cre recombinase gene (Transgenic Pedigree #1) and a responder strain, in which loxP sites (large arrowheads) have been introduced flanking critical regions, such as exon 2, of the Bmpr target gene (Transgenic Pedigree#2). Intercross matings of the two strains induces an intramolecular recombination event between the two loxP sites that excises the intervening sequences (exon 2 in this case). (B) The mating scheme used to generate mutant and normal littermates used in this study (the term ‘flox’ is used when either the floxP or floxP-neo allele could be used (see D), as both appear to function equivalently). (C) Southern blot analyses of tissues from an 18.5 dpc embryo demonstrate that the Brn4-Cre transgene efficiently mediates the rearrangement of the floxP-neo allele of the Bmpr gene (see D for the structure of this allele). In tissues derived from the neural tube, such as spinal cord, hindbrain and forebrain, efficient rearrangement of the floxP-neo allele results in a conversion of the 6.3 kb fragment to a 2.2 kb fragment; the null allele yields a 4.0 kb fragment. The small amount of Cre-mediated rearrangement in the limbs results from the ectodermally derived cells in the limbs that express the Brn4-Cre transgene (see Fig. 2); the majority of the limb derives from mesenchymal tissue that does not express the Brn4-Cre transgene. Labeled size standard (lane 1) is 1 kb DNA Ladder (Life Technologies); sizes of hybridized bands (kb) are given (left). (D) To introduce loxP sites (arrowheads) into the first and second intron, a targeting vector was engineered to contain one loxP site in the first intron, and a neor gene flanked by loxP sites in the second intron; successful targeting of this construct resulted in the floxP-neo allele depicted in this panel. The residual neor gene in the second intron of the floxP-neo allele apparently did not interfere with the function of the gene, as no discernible phenotype was detected in mice homozygous for this allele. An allele in which the neor gene was specifically removed (floxP) was generated by partial excision of the locus with Cre recombinase (Y. M., M. C. H. and R. B., unpublished). No differences in phenotype were observed whether we used the floxP-neo or the floxP allele. Box (red) above exon 3 of modified alleles corresponds to H23 probe used to genotype Bmpr pedigrees. Nh, NheI; S, SacI.
