Disruption of the Nfix gene. A) Nfix targeting vector construction and predicted PCR products. Fragments including exon 2 of the wild type Nfix gene (Wild Type Allele) were used to construct the Targeting Vector. The Targeting Vector has one loxP site inserted ~400 nt 5' to exon 2 and a FRT-flanked PGK-Neo cassette (white arrow) with a second 3' loxP site ~600 nt 3' to the end of exon 2. Arrows show the location of PCR primers within genomic DNA used to detect the Wild Type Allele (WT PCR), the targeted Conditional Allele (CA PCR) and the Knockout allele (KO PCR); and primers from outside of and within the targeting vector used to verify correct genomic targeting at the 5' (5' PCR) and 3' (3' PCR) ends of the vector. Males with the Conditional Allele were bred with females expressing Cre recombinase in oocytes from the ZP3 promoter to generate the knockout allele (After Cre) lacking exon 2. B&C) Nested PCR showing targeted integration at the 5' (B) and 3' (C) ends of the conditional allele. B) A gel of products from nested PCR reactions with primers from genomic DNA 5' to the 5' end of the targeting vector, and primers within the 5'-most loxP site that yield no product from WT DNA and a 2831 bp product (arrow) from the conditional allele (CA 5' PCR). ES clones 10, 22, 24, 25 and 38 were strongly positive while clones 27, 28, 36, 46 and 48 were negative. Marker sizes in kb are on the left. Positive signals were verified in multiple PCR reactions. C) A gel of nested PCR products of primer pairs where one primer of each pair was located in genomic DNA outside of the 3'-most fragment of DNA present in the targeting vector and the other primer was present within the 3'-most loxP site of the targeting vector (3' PCR). Negative control DNA from non-electroporated ES cells shows no signal while clones 25, 38 and 40 yield a 5096 bp product (arrow, CA 3' PCR) specific for the correctly targeted Conditional Allele. The specificity of both the 5' and 3' PCR fragments was also confirmed by restriction enzyme digestion. Marker sizes in kb are on the left. D) PCR genotyping of Nfix +/+, c/+ and c/c mice. Mice heterozygous for the CA allele were bred, tail samples of progeny were collected, and the DNA was subjected to PCR using primers that flank the 5' most loxP site yielding products of 214 bp for the wild type (WT) allele and 415 bp for the Conditional (CA) Allele. Marker sizes in bp are on the left. E) RT-PCR showing the absence of exon 2-containing Nfix transcripts in Nfix -/- mice. RNA was prepared from the livers of Nfix+/+, -/+ and -/- animals, subjected to RT reactions and the resulting cDNA was subjected to PCR with primers in exons 1 and 3 of Nfix. RNAs containing exon 2 yield products of 606 bp while RNAs lacking exon 2 yield products of 75 bp. Note that some 75 bp product is formed from RNA in WT animals and we have confirmed by QPCR and sequencing that a small fraction of Nfix transcripts in WT animals are directly spliced from exon 1 to exon 3.