Skip to main content
. 2015 Feb 4;24(10):2808–2825. doi: 10.1093/hmg/ddv042

Figure 1.

Figure 1.

Generation of a conditional Nsdhl allele. (A) Schematic diagram of the cholesterol biosynthesis pathway from lanosterol to cholesterol, with sterols listed in shaded boxes and the enzymes that catalyze each step shown next to the arrows. NSDHL, along with SC4MOL and HSD17B7, is required for the removal of two C-4 methyl groups from 4,4-dimethylcholesta-8,24-dien-3β-ol to generate zymosterol. Reduction of the C-24 double bond by DHCR24 can occur at multiple points along the pathway, but is shown only as the last step for simplicity. Ketoconazole inhibits CYP51A1 in the demethylation of lanosterol at C-14. Abbreviations: CYP51A1, cytochrome P450 lanosterol 14α-demethylase; DHCR14, 3β-hydroxysterol-Δ14-reductase; LBR, lamin B receptor; SC4MOL, sterol C-4 methyloxidase-like; NSDHL, NADH steroid dehydrogenase-like; HSD17B7, hydroxysteroid 17β-dehydrogenase 7; EBP, emopamil binding protein (3β-hydroxysteroid-Δ8,Δ7-sterol isomerase) ; SC5D, 3β-hydroxysteroid-Δ5-desaturase; DHCR7, 7-dehydrocholesterol reductase; DHCR24, 3β-hydroxysterol Δ24-reductase; T-MAS, 4,4-dimethylcholesta-8,24-dien-3β-ol. (B) Experimental strategy for generating the Nsdhlflx5 allele. The top line represents the mouse wild type Nsdhl gene from exon 4 to exon 8, indicating the position of diagnostic restrictions sites for Asp718 (A) and Bgl1 (B). The exons and introns are not drawn to scale. The position of probe sequences A and B used on Southern blots to detect homologous integration of the targeting construct are indicated. Below is a simplified map of the targeting construct generated in the vector pL451, showing loxP sites (black arrowheads) flanking Nsdhl exon 5, the neomycin-resistance gene (Neo) flanked by FRT sites (white arrowheads) for positive selection, and the thymidine kinase gene (TK) for negative selection. Homologous integration of the Nsdhlflx5 construct into the Nsdhl locus following electroporation into ES cells results in altered sizes of the Asp718 and Bgl1 restriction fragments, as shown in the third diagram. Finally, the Neo cassette was excised by FLPo-mediated recombination in ES cell clones to generate the Nsdhlflx5 allele (bottom diagram). (C) Southern blots of genomic DNA from a WT and representative Nsdhlflx5/Y neo-resistant ES clone digested with Asp718 and BglI, and hybridized with probes A and B, respectively, that lie outside of the sequence in the flx5 construct. The targeted clone showed the expected changes in size of the diagnostic restriction fragments, demonstrating homologous integration into the Nsdhl locus. (D) A Western blot of total protein prepared from E9.5 male embryos from a Nsdhlflx5/flx5 x Sox2-cre cross probed with antibodies against NSDHL (top) and β-tubulin (bottom) as a loading control. The Nsdhlflx5 control sample was from pooled cre-negative male embryos, and showed the expected 38 kDa wild type band for NSDHL. No NSDHL signal was detectable in the NsdhlΔ5 sample from pooled cre-positive male embryos. The higher level of β-tubulin signal in the NsdhlΔ5 sample is due to more total protein loaded than in the Nsdhlflx5 lane.