Figure 2.

Generation of CNS-specific Trka conditional mice. A, Trka conditional targeting strategy. Schematic representation of exons 11–17 of the wild-type mouse Trka locus; recombinant (neo⁺) and recombinant (neo⁻) alleles are also shown. LoxP sites flanked the Trka exons 12–14, whereas FRT sites flanked the neo gene, which was subsequently excised in vivo by crossing the Trka floxed mice with transgenic mice expressing Flp recombinase ubiquitously (Rodríguez et al., 2000). This resulted in the generation of the recombinant Trka^lx_neo− (Trka^lx) allele. B, Southern blot analysis of targeted ES cell clones of Trka^lx_neo+(Trka^lxn) using two probes at the 5′ and 3′ sides of the targeting construct, as indicated. BamHI restriction enzyme was used to digest the genomic DNA for the Southern blot analyses. C, PCR analysis of Trka^lxn and Trka^lx alleles. The positions of the primers used for PCR analysis are indicated in A. The PCR reaction with the primers 1 and 3, which showed successful deletion of the neo gene, was used for routine genotyping of the Trka^lx mice. D, Western blot analysis of gp140^TrkA protein in forebrains of newborn (P1) wild-type, Trka^lx/lx, and Trka^lxn/lxn mice. While TrkA protein level was nearly not detectable in forebrain lysates of Trka^lxn/lxn mice, removal of the neo cassette restored normal TrkA protein level in Trka^lx/lx mice compared to control mice. Tubulin was used to control for loading. E, Western blot analysis showing Cre-mediated removal of TrkA from P1 forebrain lysates of Trka^lx mice crossed to NestinCre mice. While TrkA protein levels in wild-type and Trka^lx/lx mice were identical, no expression of TrkA was detected in the Trka^lx/lx;NesCre^tg/+ mice (Trka^NesCre). F–K, Forebrain coronal cryosections from 1-month-old Trka^NesCre and control (Trka^lx/lx) mice were stained with antibody against TrkA. Although strongly stained neurons were found in all the indicated areas in the control mice, shown is mainly the nucleus basalis (F, G), the medial septum (H, I), and the striatum (J, K); no TrkA-positive cells were found in the Trka^NesCre mice. L–M, Transversal cryosections of the spinal chord region of E17.5 mouse stained with TrkA antibodies. Clear TrkA-positive staining is observed in neurons of the DRG in both control and Trka^NesCre mice. St, Striatum; ic, internal capsule; Gp, globus pallidus; DB-hl, diagonal band horizontal limb. Scale bars: F (for F, G), H (for H, I), 200 μm; J (for (J, K), 100 μm. B, bamHI; S, SacII; X, XhoI.