Fig. 1. Targeted disruption of the murinetn-C gene. a, Diagram of a part of the murine tn-C gene (denoted as tn-C +) covering exons 2–5, the targeting vector, the targetedtn-C gene, and the resulting mutant (denoted astn-C -) allele after Cre recombinase-mediated excisionin vitro. Artificial introduction of aBamHI site combined with the excision of the floxed exon 2 converted a 11.5 kb BamHI genomic fragment indicative of the wild-type allele (tn-C +) to a 4.6 kbBamHI genomic fragment indicative of the mutant allele (tn-C -). Selected restriction enzyme recognition sites are indicated as follows: B, BamHI;Bg, BglII; D,DraI; V, EcoRV. Probe5′A and primers 1A,1B, and 1C are indicated.b, Southern blot analysis of representative tail DNA samples from wild-type (tn-C +/+), heterozygous (tn-C +/−), and homozygous (tn-C−/−) mice. DNA was digested with BamHI and subjected to hybridization using probe 5′A (a). c, Determination of genotypes by PCR. Multiplex PCR using primers 1A,1B, and 1C (a) as performed routinely from representative tail DNA samples.d, Northern blot analysis of total RNA from wild-type (lane 1, 3) and TN-C-deficient mice (lanes 2, 4). RNA was isolated from cerebrum (lanes 1, 2) and cerebellum (lanes 3, 4) of 7-d-old mice. Wild-type samples gave rise to a broad band with a size of ∼6–8 kb. Note that the weak band in lane 4 is shifted to lower molecular mass with respect to the intensive wild-type band inlane 3. Probing blots with a GAPDH-specific probe revealed no differences in RNA amounts loaded. e, RT-PCR analysis of TN-C-specific cDNAs derived from wild-type (lanes 1, 3, 5, 7) and TN-C-deficient mice (lanes 2, 4,6, 8). Reverse transcription was performed on total RNA from cerebrum (lanes 1,2), cerebellum (lanes 3,4), lung (lanes 5,6), and thymus (lanes 7,8) of 7-d-old mice. In the subsequent PCR, primers were used as depicted. Note that the mutant TN-C message lacking exon 2 could be amplified from all TN-C-deficient tissues tested.