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. 2014 Dec 30;24(8):2163–2174. doi: 10.1093/hmg/ddu736

Figure 1.

Figure 1.

Generation of Nefl P8R and N98S knock-in mice. (A) Strategy for generating the knock-in mice: a targeting vector containing the point mutation in the first exon of Nefl, and a Loxp-Neo-Loxp cassette inserted in the first intron was constructed and used to generate the knock-in mice. The targeted allele could be differentiated from the WT allele by PCR using the 4F and 4R primers. (B) Ethidium bromide-stained agarose gel of PCR analyses of the F1 mice showing the WT and targeted (L83 (N98S)) alleles; five of the nine animals showed both alleles, the other four were WT. (C) RNA was isolated from brains and spinal cords of Nefl+/+, NeflP8R/+ NeflP8R/P8R and NeflN98S/+ mice and the amount of NFL mRNA was quantified by RT–PCR and compared with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. Ratios of NFL mRNA and GAPDH mRNA for the different genotypes are shown. Experiments were repeated with three sets of animals. (D) Western blots of brain, spinal cord and sciatic nerve from Nefl+/+, NeflP8R/+, NeflP8R/P8R and NeflN98S/+ probed with anti-NFL-N antibody and anti-GAPDH. (E) Ratios of NFL/GAPDH from the western blots shown in D. (Brain: Nefl+/+: n = 10; NeflP8R/+: n = 9; NeflP8R/P8R: n = 10; NeflN98S/+: n = 9; Spinal cord: Nefl+/+: n = 10; NeflP8R/+: n = 9; NeflP8R/P8R: n = 10; NeflN98S/+: n = 9; Sciatic nerve: Nefl+/+: n = 9; NeflP8R/+: n = 8; NeflP8R/P8R: n = 9; NeflN98S/+: n = 7). Significance was calculated using a one-tailed, type 3 t-test in excel (*P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001).