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. 2005 Mar 11;102(12):4554–4559. doi: 10.1073/pnas.0406380102

Fig. 1.

Fig. 1.

smpd3-knockout strategy, genotype analysis, and absence of smpd3 expression. (A) Targeted disruption of the smpd3 locus. (a) wt murine smpd3 gene locus. (b) Targeting construct. (c) Targeted smpd3 locus. (B) Southern blot hybridization analysis. SstI-restriction fragment length polymorphisms of DNA of ES clones and tail biopsies of control (+/+), smpd3+/–, and smpd3–/– siblings of F1 and F2 generation. Diagnostic fragments: wt DNA, 4.6 kb; and smpd3–/–, 6.4 kb. (D) Northern blot hybridization of total brain RNA of +/+, +/–, and –/– of 3-wk-old mice. Probe: smpd3 cDNA, β-actin cDNA loading control. (E) Western blot analysis of brain protein extract (p14). (C) Genotyping of smpd2–/–smpd3–/– double mutant by PCR. Tail DNA from wt, smpd2±smpd3–/–, and smpd2–/–smpd3–/– mice was used as template for PCR with primer pairs listed under Methods and Materials. Lanes: a, wt smpd2 sibling; b, smpd2+/–; c, smpd2–/–. The 3-kb fragment indicates the wt–, the 3.9-kb fragment, the targeted smpd2 allele. Lanes: a′, PCR fragment of the smpd3+/+ and c′, the smpd3–/– allele. The 1.3-kb fragment monitors the wt smpd3+/+ allele, the 2.9-kb fragment, and the smpd3–/– allele. (D) Northern blot analysis. (E) Western blot analysis.