OLs express R-Ras1 and R-Ras2 in the main myelinated tracts of the CNS. A, Immunoblot of adult optic nerve lysates showing R-Ras1, R-Ras2, and R-Ras3 expression. Two different antibodies against R-Ras1 or R-Ras2 recognized both proteins, a band of 25 kDa corresponding to R-Ras1 and another of 21 kDa corresponding to R-Ras2. No bands were observed in optic nerve lysates from R-Ras1^−/−;R-Ras2^−/− mice. R-Ras3 expression was not modified in R-Ras1^−/− and/or R-Ras2^−/− showing a band of 24 kDa (n = 3). B, C, Western blot of corpus callosum (B) and spinal cord (C) extracts from control mice confirmed expression of R-Ras1, R-Ras2, and R-Ras3 in primary CNS myelinated tracts (n = 3). D, Schematic representation of the targeting strategy to generate the R-Ras1-null mutant. A Neo cassette flanked by FRT sites was inserted into intron 1 together with two loxP sites flanking exons 2 and 6. The positions of the primers designed for genotyping are shown by the arrows. E, PCR genotyping. A mixture of primer 1 and primer 2 detected the R-Ras1^+/+ (∼100 bp) allele by PCR and a mixture of primer 3 and primer 4 detected the R-Ras1^−/− target allele (∼1000 bp). F, Generation of R-Ras2^−/− mice. Shown is an illustration of the retroviral insertion site of the R-Ras2 gene in intron 1 and the positions of the three primers used for genotyping. G, Analysis of the relative expression of R-Ras1 and R-Ras2 by qRT-PCR in control, R-Ras1^−/−, R-Ras2^−/−, and R-Ras1^−/−;R-Ras2^−/− adult mice normalizing all data to control mice. Values are expressed as the mean fold change of 3 different animals, p < 0.0001. H, Longitudinal optic nerve sections from control, R-Ras1^−/−, R-Ras2^−/−, and R-Ras1^−/−;R-Ras2^−/− adult mice showing colocalization between R-Ras1 (top) and R-Ras2 (bottom, green) with Nkx2.2, an oligodendroglial cell marker (red). No staining for R-Ras1 or R-Ras2 was observed in the double-mutant mice (n = 3). All samples were obtained from adult mice (P90). Scale bar, 25 μm.