Materials and Methods Supporting Figures S1-s7 and Legends Supporting References Materials and Methods

Targeting construct, genotyping, colony generation and analysis. We have isolated Nobox genomic clones from a 129S6/SvEv genomic library and generated a targeting construct (Fig. S1A). The Nobox targeting construct was electroporated into AB2.2 ES cells to mutate the wild type Nobox locus by homologous recombination as previously described(S1,S 2). The mutant Nobox allele replaces exons 2-8 with the Pgk1-HPRT cassette. We screened 192 ES clones by Southern blot analysis and identified 19 ES cells that carry a targeted mutant allele by Southern blot analysis. The Nobox +/-ES cells were injected into blastocysts, and chimeras were generated from three independent ES cell lines. The Nobox +/-mice generated from chimeras were bred to produce homozygous null Nobox-/-mice, which were identified by Southern blot analysis of tail DNA (Fig. S1, B, C and D). Mice were genotyped by Southern blot analysis and multiplex PCR (primers and conditions are available from A.R. on request).