Fig. 1. Generation of Ebf2-null mice by homologous recombination. (A) The wild type Ebf2 locus (first six exons only), targeting construct and targeted Ebf2 locus. Letters represent restriction sites. A, ApaI; E, EcoRI; H, HindIII; S, SalI; X, XhoI; solid boxes represent exons; stripes represent introns; gray boxes represent genomic sequences used to generate 5' and 3' probes; triangles represent loxP sites; the PgkNeo, PgkTk minigenes and the lacZ cDNA are represented as empty boxes. (B) Restriction patterns obtained by hybridizing EcoRI-digested DNAs form parental ES cells (par) and homologous recombinant clones (rec) with the 5' probe, and HindIII-digested DNAs with the 3' probe. Fragments corresponding to the recombinant locus are 3.5 kb in EcoRI digests and 9 kb in HindIII digests. (C) Restriction patterns obtained by hybridizing with the 3' probe HindIII-digested DNAs from wild type (+/+), heterozygous (+/-) and homozygous mutant (-/-) mice. (D) RT-PCR experiments conducted starting from total RNA from E13 embryos. Lanes 1,2, wild-type RNAs; lanes 3,4, Ebf2^-/- RNAs; lanes 1,3, RT+ experiments; lanes 2,4, RT—controls; lane 5, distilled H₂O was used as template for the PCR reaction (blank). A Gapdh RT-PCR product was used for normalization. The 350 Ebf2 cDNA fragment failed to amplify from Ebf2^-/- reverse transcription reactions. (E) General appearance of two 20-day-old F₂ Ebf2^-/- male mice (-/-) compared with one wild-type F₂ male littermate (+/+).