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Fig. 1 | BMC Biology

Fig. 1

From: Sudden death due to paralysis and synaptic and behavioral deficits when Hip14/Zdhhc17 is deleted in adult mice

Fig. 1

Generation of Hip14 conditional knockout mice. The targeting vector that was used is shown in (a). It was generated using PCR cloning of the 5′ and 3′ homology arms (5.5 and 3.2 kbp, respectively) and the deletion region (conditional knockout region [cKO]) with the indicated restriction enzyme sites added by PCR and used for cloning into the targeting vector, such that loxP sites are oriented in the same direction up and downstream of the cKO region. The deletion region, in gray, includes exon 2 and upstream and downstream intronic sequences to a total of 1.1 kbp. The targeting vector also includes a positive neomycin (Neo) selection cassette flanked by flippase recognition target (frt) sites and a negative diphtheria toxin A (DTA) selection cassette outside of the homology arms. The wild-type (WT) allele is shown in (b) with the 5′ and 3′ homology arms and the cKO region indicated. The recombined allele (Hip14 F) is shown in (c). The neo cassette was removed during embryonic cell culture after targeting by electroporation with Flp recombinase and negative selection with G418. The knockout allele following expression of Cre recombinase is shown in (d) where recombination between the loxP sites occurs by the action of Cre leading to deletion of the cKO region, including exon 2. This deletion results in a frameshift mutation and multiple premature stop codons. The expression of Hip14 mRNA in the striatum, hippocampus, cortex, cerebellum, spleen, liver, kidney, and heart in iHip14 Δ/Δ mice and in iHip14 F/F control mice relative to β-actin is shown in (e) (N = 3). There was a 90% or greater decrease in Hip14 mRNA in iHip14 Δ/Δ mice compared to control mice. f HIP14 protein expression in the striatum, hippocampus, cerebellum, and cortex from iHip14 Δ/Δ and iHip14 F/F mice, where the tissues were collected 10 days post-TM treatment, is shown. Also shown is whole brain collected 6 weeks post-TM treatment from iHip14 Δ/Δ and iHip14 F/F mice. Whole cell lysate was run on western blot and probed with anti-HIP14 and anti-β-tubulin antibodies. The HIP14 immunoblot is the top panel in each set of images; the β-tubulin is in the bottom panel. The amount of HIP14 protein expressed is quantified in the graph relative to β-tubulin expression (N = 2–10). A 90% or greater decrease in HIP14 was observed in iHip14 Δ/Δ mice. Data were analyzed using Student’s t test

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