Figure 1
From: Enhanced neuronal Met signalling levels in ALS mice delay disease onset
Temporal and tissue-specific Met transgene activation using Cre-mediated recombination. (a) The R26LacZ−stop−Met construct targeted in the Rosa26 locus is shown. The targeting construct consists of the CMV-enhancer and the chicken β-actin promoter (CMV/β-actin) driving the β-geo reporter and a Met chimeric cDNA. The reporter gene is followed by three copies of the SV40 polyadenylation signal (3xpA) and flanked by loxP sites. The Met chimeric cDNA consists of a 5′ portion encoding the mouse extracellular domain fused to a 3′ region coding for the intracellular human portion. Positions of the probes used for Southern analysis (a and b) as well as primers used for PCR (1, 2, 3) are indicated. (b) Southern blots of neo-resistant ES clones analyzed with probe a (EcoRI digestion; top) and probe b (EcoRV digestion; bottom). The 15.5 Kb band corresponds to the wild-type allele whereas the 5.5 Kb band depicts the recombinant allele in clones 1 and 2. (c) PCR of mouse-tail genomic DNAs showing the mutant allele before and after Cre recombination (top). The presence of an amplicon in the Nes-R26Met mice with the 1+2 primers is detected because genotypes are performed using DNA extracted from tails, which include tissues with and without recombination. PCR showing the genotypes of heterozygous and homozygous Nes-R26Met mice (bottom). (d) Western blots showing the expression of the Met chimeric protein (using anti-human Met antibodies) in brain and spinal cord extracts from embryos (E15.5) and newborn mice (P7) with the indicated genotypes. (e) Quantification analysis of Met chimeric protein levels versus endogenous Met. Western blot analysis of protein extracts from dissected brains and spinal cords at different developmental stages using antibodies recognizing the Met kinase domain (MetKD). (f) Quantification analyses revealed that levels of Met chimeric proteins were at least five- and sevenfolds increased in brains and spinal cords of heterozygous Nes-R26Met, respectively, when compared with the endogenous mouse Met. Met chimeric levels were double in homozygous mice. Numbers on columns indicate fold of increase in Met protein levels over the endogenous Met. In the graph, Nes-R26Met and Nes-R26Met/Met genotypes are indicated as M/+ and M/M, respectively. Values are expressed as means±S.E.M. Tub, tubulin; hMet, human-Met
