Figure 1. Construct design and validation.

A) The genome region around PREPL's exon 11 was used to design a targeting vector for homologous recombination. This vector included a neomycin (NEO) cassette for positive selection, Frt sites flanking the NEO cassette, LoxP sites flanking exon 11, a HindIII restriction site for 5′-Southern blots, and an NdeI site for 3′-Southern blots. Homologous recombination in ES cells generated cells carrying recombinant genomic DNA, which according to standard nomenclature (Gene^tm#Labcode) is referred to as Prepl^tm1Sagh. These ES cells were then used to generate chimeric mice designated Prepl^+/tm1Sagh. B) The germ line transmission of the Prepl^tm1Sagh allele to the offspring from chimera-C57BL/6J crosses was confirmed by Southern blots of tail genomic DNA. Restriction digests of the genomic DNA with HindIII (5′) or NdeI (3′), followed by probing with a 5′ or 3′ specific probe generated a single band for Prepl^+/+ (left lane), whereas an additional, lower molecular weight band is generated in mice carrying the Prepl^tm1Sagh allele (right lane). C) Prepl^+/tm1Sagh were then crossed with mice expressing Flp recombinase to produce Prepl^+/tm1.1Sagh mice (middle) lacking the NEO cassette, and finally crossed with mice ubiquitously expressing Cre recombinase, resulting in Prepl^+/tm1.2Sagh mice (bottom) which lack exon 11. We refer to these Prepl^+/tm1.2Sagh mice as PREPL^+/− mice. D) Representative PCR genotyping results for PREPL^+/+, PREPL^−/− and PREPL^+/− mice. Using this strategy, one can easily distinguish these three genotypes as well as other possible genotypes, such as the Prepl^tm1.1Sagh allele.