Fig. 1. Generation of a Npnt-null allele. (A-F) Targeting strategy for generating Npnt mutant alleles using a BAC containing part of the Npnt locus. (A) Representation of the modified BAC (273P10) used for targeting, showing the first 11 exons (vertical bars) of the Npnt gene present in this BAC. Boxed region spans exons 1 and 2. (B,C) Illustration of the modifications that were made to the BAC DNA, including insertion of loxP sites in the introns 5' and 3' to the first exon, an insertion of a neomycin expression cassette flanked by frt sites, and restriction sites (asterisk). (D) Representation of the Npnt^floxneo allele, produced following homologous recombination between the modified BAC and the Npnt locus in ES cells. (E) Mice carrying Npnt^floxneo were crossed to mice expressing CRE recombinase under the ß-actin promoter (Lewandoski et al., 1997) to create the Npnt^ex1 allele. Note that this allele still contains the neo cassette. (F) Southern blot of DNA from two ES cell clones, one heterozygous for the Npnt^floxneo allele and the other wild-type at the Npnt locus. An EcoRI digest produces an 8 kb wild-type and a 4 kb mutant band. Each clone is represented by a series of three fourfold dilutions (left to right). (G) RT-PCR for Npnt and Gapdh expression in Npnt^+/+, Npnt^+/- and Npnt^-/- mice. Total RNA was extracted from spleens of newborn mice using the RNeasy mini kit (Qiagen Inc., Valencia, CA), and reverse transcribed using Superscript II and oligo(dT)12-18 Primer (Invitrogen Corp., Carlsbad, CA). PCR was performed using forward and reverse primers that recognize sequences in Npnt exons 4 and 8, respectively, and primers that recognize a sequence in Gapdh exon 3. Control reactions without reverse transcriptase were negative for both PCR reactions (not shown). (H) Immunostain for nephronectin in kidneys from wild-type and Npnt^ex1 homozygous (null) newborn mice using an anti-nephronectin antibody that recognizes sequences in the C-terminal region of the protein (Brandenberger et al., 2001). Primers for wild-type allele, Npnt^WT, NN-1A: 5'-AGTCCATCCTGATCACTGGCT-3' and NN-1C: 5'-GCAACCTTCAGCGTCCC-3', band size 279 bp. Primers for mutant allele, Npnt^ex1, NN-1B: 5'-TATGGCTTCTGAGGCGGAAAGAAC-3' and NN-1F: 5'-AAGTGGAGCTTCAGGACACAG-3', band size 509 bp.