Generation of floxed Mfrn1 alleles. Schematic description of the wild-type Mfrn1 locus (A) and the locus after introduction of a loxP-FRT-neomycin resistance-FRT cassette in intron 1 and an additional loxP site between exons 2 and 3 (B). The position of the 5′-flanking probe used for Southern blot analysis is shown as a blue bar in panels A and B. (C) Flp recombinase–mediated excision in which the neomycin cassette was removed by FRT recombination but the coding sequence was left intact. (D) Complete Cre recombinase–mediated excision of exon 2 to inactivate the Mfrn1 gene. For panels A through D, LoxP sites are shown as red triangles and FRT sites are shown as green diamonds. (E) Southern blot analysis of the 5′ end of the locus in ES cells after BamHI digestion. Lane 1 shows the wild-type allele at 7.4 kb, and lanes 2-4 targeted recombination with a wild-type allele at 7.4 kb and a recombinant allele at 4.5 kb, as indicated in panels A and B. (F) PCR analysis of the offspring of Mfrn1^floxneo/+ and Mfrn1^+/+ chimeric mice. PCR was done using primers F, R1, and R2, as indicated in panels A and B. Lane 1 and lane 4 show the Mfrn1flox^neo/+ genotype and lanes 2 and 3 the Mfrn1^+/+ genotype. The presence of Neo (primers F × R1) gives a product of 120 bp; the absence of Neo (primers F × R2) gives a product of 60 bp. (G) PCR analysis of Cre recombinase–mediated excision of exon 2. PCR was done using primers F, R3, and R4, as indicated in panels C and D. The wild-type allele gives a product of 173 bp (primers F × R3), the flox allele gives a product of 273 bp (primers F × R3), the negative allele gives a product of 428 bp (primers F × R4), and R3 does not hybridize anymore. Lane 1 shows the Mfrn1^flox/− genotype, lane 2 shows the Mfrn1^+/− genotype, lanes 3 and 4 show the Mfrn1^flox/+ genotype, and lane 5 shows the Mfrn1^+/+ genotype.