7-Dehydrocholesterol–dependent proteolysis of HMG-CoA reductase suppresses sterol biosynthesis in a mouse model of Smith-Lemli-Opitz/RSH syndrome
J. Clin. Invest. Barbara U. Fitzky, et al. 108:905 doi:10.1172/JCI12103 [Go to this article.]

Figure 1
(a) Disruption of the Dhcr7 gene. The coding sequence of exon 8 and the 5′ flanking splice acceptor site and part of the 3′ flanking untranslated region (indicated in gray) of the wild-type gene were replaced with a neomycin resistance gene in opposite orientation to Dhcr7 transcription. Restriction sites used for subcloning of short and long Dhcr7 gene segments are shown. The targeting construct was linearized with ClaI (arrow). Primers employed for PCR genotyping are labeled a, b, and neo (see Methods). (b) Tentative topology model of Dhcr7. The induced Dhcr7 mutation is expected to delete one-third of the Dhcr7 protein (the residues shown as filled circles). The box indicates the putative sterol-binding site REF. (c) Total RNA extracted from the livers of Dhcr7–/–, Dhcr7+/–, and wild-type newborn mice was reverse transcribed and amplified using specific primers for the 3′-end of Dhcr7 mRNA and GADPH. (d) Immunoblot analysis of Dhcr7. Upper panel: Loading decreasing amounts of total hepatic protein results in corresponding reductions in Dhcr7 Ab response. Lower panel: Microsomal protein from yeast (WA0) and human tsA-201 cells (tsA) heterologously expressing DHCR7 with an N-terminal myc tag (15) and from livers of newborn wild-type mice demonstrated immunoreactive Dhcr7 protein. Bands corresponding to myc-tagged (myc) and wild-type (WT) DHCR7/Dhcr7 proteins, respectively, are indicated by arrowheads. In contrast, no immunoreactive band corresponding to wild-type Dhcr7 was detected in 40 μg of microsomal protein from either homozygous mice (last two lanes) or mock-transfected yeast (MOCK).