Fig. 6. Construction of transgenic NPD mice expressing the R496L and ΔR608 ASM mutations.

(A) Schematic diagram of the mouse SMPD1 promoter region (mSMPD1p). The 1.6 kb Acc I fragment was cloned into the pGL3 Basic vector upstream of a luciferase cassette (pGL3 mSMPD1p). This fragment also was cloned into the pNEB193 vector upstream of cDNAs encoding human ASM harboring the R496L and ΔR608 mutations. An SV40 polyadenylation signal was introduced downstream (Transgenic Constructs). (B) The pGL3 Basic and pGL3 mSMPD1p vectors were transfected into HEK293T17 cells. Cell extracts were prepared and the in vitro luciferase activity was measured. Data are presented as means ± SD (n = 3). (C) Transgene constructs were microinjected into one-cell stage embryos derived from superovulation of ASM knock-out mice and individual founder mice carrying the R496L and ΔR608 transgenes were identified. PCR genotyping of representative F2 offspring from each founder line is shown. (D). Northern blot analysis of total RNA prepared from liver (L) and brain (B) of wild-type (WT), ASM knockout (KO) and representative F2 transgenic mice from each founder line (R496L and ΔR608). Note that the probe used was derived from the SV40 polyadenylation sequence, and thus only the transgenes were detected. (E). Livers and brains were harvested from wild-type (WT), ASM knockout (KO) and several F2 transgenic mice (Tg) for each mutation, and the in vitro ASM activities were measured. All reactions contained 1–10 µg of protein and 100 µM B12SM substrate (final concentration). Data are presented as percentage means ± SD (n = 3). The value calculated for wild-type mice was set as 100 %. Note that the mean activity values in the brains of the ΔR608 transgenic animals were significantly greater (P < 0.01) than the ASMKO background.