FIG. 1.

Allelic recombination reporter for homologous chromosomes. (A) Allelic recombination substrates S2neo and Pneo were sequentially targeted to the 129/Sv and BALB/c alleles, respectively, of the Rb locus in the mouse ES cell line v17.2. Southern blot hybridization was performed with the indicated Rb probe fragment, which is located outside the targeting fragments, for detection of wild-type and targeted alleles from an EcoRI digest of genomic DNA. Targeting with either S2neo or Pneo gives rise to an EcoRI fragment of the same size. The neo genes are inserted upstream of exon 19 (vertical black bar) at a site which appears to be nondisrupting for Rb expression (35). (B) Both targeting events used a linked hygromycin resistance (hyg) gene (to select for chromosomal integration) which was removed after each targeting event by Cre-mediated recombination between the loxP sites (open circles) flanking the hyg genes. The alleles are indicated with an H in cases in which they contain a hyg gene (SH, 129/S2neo-hyg; PH, BALB/c/Pneo-hyg) and without an H in cases in which the hyg gene has been removed (S, 129/S2neo; P, BALB/c/Pneo). Removal of the hyg gene was confirmed by Southern blotting (using the neo probe fragment depicted in the diagram) with a StuI/HindIII digest of genomic DNA. Using the indicated StuI and HindIII polymorphisms, this analysis also confirms linkage of the S2neo gene to the 129 allele. Southern blot signals for cell lines with the genotypes SH/+, S/+, S/PH, and S/P, where + indicates the nontargeted allele, are shown. St, StuI; P, PacI; HIII, HindIII; N, NcoI, RI, EcoRI, Ea, EagI.