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. 2015 Oct 28;35(43):14501–14516. doi: 10.1523/JNEUROSCI.1056-15.2015

Figure 1.

Figure 1.

Generation and characterization of Nat8L-deficient mice. A, Targeting strategy to generate a conditional, floxed Nat8L allele. An FRT-flanked neomycin resistance gene and a lacZ reporter gene with an upstream splice acceptor site and an internal ribosome entry site (IRES) were inserted downstream of exon 3. Mice containing the targeted floxed Nat8L allele were crossed with transgenic deleter-Flp mice to remove the neomycin selection cassette or with pgk-Cre transgenic mice to create a Nat8L knock-out allele expressing the lacZ reporter gene under the control of the Nat8L promoter. The position of restriction enzyme cut sites used for Southern blot analysis (H, HindIII; M, MfeI) and the position of primers (p1–p3) used for PCR are indicated. B, Southern blot analysis of mouse genomic DNA digested with HindIII of wild-type (+/+) and heterozygous (+/−) Nat8L mice. PCR analysis of genomic DNA from Nat8L+/+, Nat8L+/−, Nat8L−/−, and heterozygous floxed (fl/+) mice: wild-type allele = 225 bp; knock-out allele = 266 bp; floxed allele = 316 bp (position of PCR primers are shown in A). C, Northern blot analysis of Nat8L expression in brain of 11- and 20-d-old Nat8L−/−, Nat8L+/−, and Nat8L+/+ littermates indicating the absence and reduced expression of Nat8L mRNA in Nat8L−/− and Nat8L+/− mice, respectively, and upregulation of Nat8L during postnatal development in wild-type mice. Two different mRNAs are present because of an alternative polyadenylation site in exon 3. D, NAA synthase activity was undetectable in Nat8L−/− mice and reduced to ∼60% in Nat8L+/− mice compared with Nat8L+/+ littermates (age: 2 months; mean ± SD, n = 3; *p < 0.05, t test). E, Quantitative real-time PCR revealed no significant changes in the expression of Aspa, ATP:citrate lyase, PLP, and MBP in total brain of 20-d-old Nat8L−/−, Nat8L+/− and wild-type littermates (mean ± SEM, n = 3). F, Myelin was isolated from the brains of 2-month-old Nat8L+/+ and Nat8L−/− mice by sucrose gradient centrifugation. No significant difference in myelin content was observed (mean ± SD, n = 3). G, MBP immunofluorescence staining on brain paraffin sections of 1-year-old animals showed no obvious difference between wild-type and Nat8L−/− mice in cerebellum (G) or other brain regions (data not shown). Scale bar, 200 μm.