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. 2015 Jul 3;78(3):426–438. doi: 10.1002/ana.24453

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© 2015 The Authors Annals of Neurology published by Wiley Periodicals, Inc. on behalf of American Neurological Association.

This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

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Expression of C9orf72 and generation of C9orf72 conditional knockout mice. (A–G) In situ hybridization shows expression of C9orf72 in coronal sections of the adult brain and spinal cord. (F, G) Double staining combining in situ hybridization for C9orf72 and immunohistochemistry for SMI132, a marker for motor neurons (indicated by arrowheads). (E, M) Sections incubated with sense probes do not display specific staining. (H–O) At embryonic and early postnatal stages, C9orf72 is expressed in spinal cord motor neurons (arrowheads in H–J), the retina, olfactory system, and other brain regions, in addition to non‐neuronal tissues such as kidney. (P) Schematic representation of the C9orf72 targeting vector, targeted allele, floxed allele following Flp recombination, and deleted allele following Cre recombination. Black arrows indicate transcription start site, gray arrows indicate location of loxP primers (P1, P2), and numbers indicate exons. Please note that exons 4 and 5 are shared by all 3 reported mouse C9orf72 isoforms. (Q) Genotyping using loxP primers on DNA isolated from ear and brain tissue of Nestin‐Cre^+/−;C9orf72^+/+ and Nestin‐Cre^+/−;C9orf72^fl/fl mice shows brain‐specific recombination. (R) Quantitative real‐time polymerase chain reaction shows tissue‐specific reduction of C9orf72 levels in Nestin‐Cre^+/−;C9orf72^fl/+ and/or Nestin‐Cre^+/−;C9orf72^fl/fl in brain (0.493, p = 0.0004 and 0.044, p = 0.00007) and spinal cord (0.684, p = 0.083 and 0.145, p = 0.002), but not in muscle (0.789, p = 0.594 and 0.585, p = 0.169) or liver (1.94, p = 0.219 and 1.74, p = 0.473). Results are shown with Tbp as a housekeeping gene. Data are presented as mean ± standard deviation, n = 3 for each genotype. (S) In situ hybridization for C9orf72 shows loss of C9orf72 expression in spinal cord of Nestin‐Cre^+/−;C9orf72^fl/fl mice. **p < 0.01, ***p < 0.001. CA1 = cornu ammonis 1; Cx = cortex; DG = dentate gyrus; DRG = dorsal root ganglia; F = Frt site; GL = granular cell layer; GM = gray matter; L = loxP site; Mol = molecular layer; N = neomycin cassette; OE = olfactory epithelium; P1 = forward loxP primer; P2 = reverse loxP primer; Ret = retina; RN = red nucleus; VNO = vomeronasal organ; WM = white matter; WT = wild type. Scale bars: A, 200μm; H, 100μm; K, 100μm, S, 150μm.