Figure 1

Conditional deletion of the fdft1 gene in vivo. (A-F) Expression of cre-recombinase in transgenic mice (Tg(mα6-cre)B1LFR) is illustrated by expression of yellow fluorescent protein (YFP) from the R26R-EYFP reporter allele. Vibratome sections of adult cerebellum were double-labeled for the Bergmann glia marker S100β (in A) and the recombination marker YFP (in B). Purkinje neurons (P) and interneurons in the molecular layer (ml) are identified as unstained "holes" (in A-C). YFP is exclusively expressed in granule cells of the granule cell layer (gl) and in their axons that form a weakly stained "lawn" of parallel fibers in the molecular layer. The merge in (C) highlights the lack of YFP expression in Bergmann glia (in red, arrow head). A single confocal plane is depicted, scale bar = 100 μm. (D-E) Overview of targeted cells in a triple transgenic mutant mouse (fdft1^flox/flox, Tg(mα6-cre)B1LFR, YFP-reporter). Staining vibratome sections from an adult animal for YFP reveals that cerebellar granule cells, pontine nuclei (white arrow head in E) and reticulotegmental nucleus of the pons (black arrow head in E) survive into adulthood in the absence of squalene synthase. The YFP-positive axons from the targeted pontine neurons enter the cerebellum via the middle cerebellar peduncle (arrowheads in E) and blowup (in F). scale bar = 1 mm (in D-E) and 50 μm (in F). (G) 80 % of all cells in the cerebellum are recombined. Southern blot on total cerebellar DNA from a control (con) and mutant (mut) animals, age two months, was hybridized to a probe that recognizes distinct fragment sizes for the flox allele (flox) and the recombined null allele (null). (F) The level of squalene synthase protein is not significantly reduced in vivo at postnatal day 16. A western blot of total cerebellar lysate (cerebellum) or the pontine nuclei from two controls (con) and two mutants (mut) was probed for squalene synthase (SQS) and glyceraldeyde-3-phosphate dehydrogenase (GAPDH) as a loading control. Apparent molecular weights are indicated on the right. Squalene synthase was consistently detected as a doublet in the pontine nuclei, but not in cerebellum or cortex (data not shown).