Skip to main content
. 2003 Jul 28;100(16):9566–9571. doi: 10.1073/pnas.1633579100

Fig. 6.

Fig. 6.

Targeted inactivation of the mouse nNOS gene. (A) Targeting strategy. (a) ORF of the nNOS gene. Exons are numbered, and regions encoding structural domains and cofactor-binding sites are shown. (b) Structural organization of the nNOS locus (according to GenBank accession no. NT_039312). (c) Structure of WT allele, targeting construct, and targeted KOex6 allele. Thick vertical bars, exons; thin vertical lines, restriction enzymes sites (A, ApaI; E, EcoRI; N, NheI; P, PmlI); arrows, the directions of transcription of the neomycin phosphotransferase gene (NEO) and the thymidine kinase gene (TK); thick horizontal bars, the 3′ and the 5′ hybridization probes used for Southern blot analysis; thin horizontal lines with arrowheads, ApaI and NheI fragments detected with 3′ and 5′ probes, respectively. (d) Southern blot analysis of mouse tail DNA. The 5.3- and 6.5-kb NheI fragments detected with the 5′ probe correspond to the WT allele and to the properly targeted KOex6 allele, respectively. The 12.0- and 8.2-kb ApaI fragments detected with the 3′ probe correspond to the WT allele and to the properly targeted KOex6 allele, respectively. (e) PCR analysis of mouse tail DNA with two pairs of primers for detection of the WT allele (607-bp fragment) and the KOex6 allele (468-bp fragment). (d and e) The sample genotype is indicated above blot or gel. (B) Removal of exon 6 results in complete inactivation of the nNOS gene. (a) RT-PCR analysis of total RNA isolated from whole mouse brains. A pair of primers used detects the 661-bp cDNA product of the WT nNOS allele (+/+,+/-), which is absent in KOex6(-/-) RNA sample. (b) Western blot analysis of whole mouse brain protein extracts. Blots were probed with antibodies specific either to the amino terminus (N-term) or carboxyl terminus (C-term) of the nNOS. A 160-kDa protein band corresponding to the nNOS was not detected in KOex6 (-/-) brain sample. Note that expression levels of nNOS enzyme in WT brains were approximately two times higher than in heterozygous brains. (c) NOS catalytic activity in whole brain protein extracts. Note that enzymatic activity in KOex6 brain homogenates was at background levels (0.15% of that in WT). NOS activity in heterozygous brains was 44.2%, approximately half that of WT, which correlates with expression levels of the nNOS in WT and nNOS+/- brains according to immunoblotting data (Fig. 6Bb). Taken together these results suggest that there is no nNOS allelic compensation in mice heterozygous for the KOex6 mutation. (d–f) The absence of nNOS in the brains of adult KOex6 mice is evident after diaphorase staining of the whole brain (d) and the OB (e) and by immunohistochemistry in the OB with anti-nNOS antibody (f). (e and f) Higher-power images of the regions indicated by the boxes in d. (Scale bars: d, 1 mm; e–f, 100 μm.) In a–f, the sample genotype is indicated.