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, 48 (10), 596-602

Hoxb8-Cre Mice: A Tool for Brain-Sparing Conditional Gene Deletion

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Hoxb8-Cre Mice: A Tool for Brain-Sparing Conditional Gene Deletion

Robert Witschi et al. Genesis.

Abstract

The spinal cord is the first site of temporal and spatial integration of nociceptive signals in the pain pathway. Neuroplastic changes occurring at this site contribute critically to various chronic pain syndromes. Gene targeting in mice has generated important insights into these processes. However, the analysis of constitutive (global) gene-deficient mice is often hampered by confounding effects arising from supraspinal sites. Here, we describe a novel Cre mouse line that expresses the Cre recombinase under the transcriptional control of the Hoxb8 gene. Within the neural axis of these mice, Hoxb8-Cre expression is found in spinal cord neurons and glial cells, and in virtually all neurons of the dorsal root ganglia, but spares the brain apart from a few cells in the spinal trigeminal nucleus. The Hoxb8-Cre mouse line should be a valuable new tool for the in vivo analysis of peripheral and spinal gene functions in pain pathways.

Figures

Fig. 1
Fig. 1. Generation of Hoxb8-Cre mice
(A) Transgene construct and respective genomic context in the murine locus. Red bars indicate coding regions of exons (X). Between Hoxb9 and Hoxb8 genes an artificial sequence (blue letters) containing a starting ATG codon was inserted. (B) Hoxb8-Cre-induced lacZ activity in E9.5 and E11.5 embryos. (C) Southern blot of EcoRV digested genomic liver DNA from a Hoxb8-Cre transgenic mouse (line 1403) hybridized with a probe against the Hoxb8 promoter. A hybridization intensity ratio of about 0.6 between Hoxb8Cre transgene (1.3 kb) and Hoxb8 wild-type (4.4 kb) suggests the presence of a single copy of the transgene.
Fig. 2
Fig. 2. lacZ activity in co-transgenic Hoxb8-Cretg+/R26R mice in neural tissue
(A) Coronal section of the spinal cord at lumbar segment L2. Dorsal horn (dh); ventral horn (vh); white matter (wm). (B) Horizontal section of the upper cervical spinal cord and cerebellum (cb) showing a gradual decrease of lacZ activity towards more anterior cervical segments. (C) Sagittal brain section with no visible Hoxb8-Cre-induced lacZ activity. (D) Sagittal section including brainstem, spinal trigeminal nucleus (Sp5C), and cerebellum (cb). (E) Lumbar DRG. All sections were obtained from 6 – 7 week old mice, incubated with X-Gal, and counterstained with acidified hematoxylin.
Fig. 3
Fig. 3. Histochemical analysis of co-transgenic progeny of Hoxb8-Cre mice crossed with R26R and RA/EG reporter strains
(A, B) Neuronal expression. (A) Coronal thoracic spinal cord section from a co-transgenic Hoxb8-Cretg+/R26R mouse. β–gal (Alexa Fluor488; left panel) and NeuN (Cy3; right panel) immunofluorescence on the same section. Scale bar: 100 μm. (B) Confocal immunofluorescence analysis of a coronal section of the lumbar spinal dorsal horn of a co-transgenic Hoxb8-Cretg+/R26R mouse. Superposition of 15 images taken at 0.3 μm intervals. β-gal (Alexa Fluor488), NeuN (Cy3) and merged view. Arrows indicate β-gal immunoreactive granula in close association with NeuN positive structures. Scale bar, 20 μm. (C) Glial expression analysis. Coronal section from the lumbar spinal cord of Hoxb8-GlyT1−/− mice and Hoxb8-Cre negative wild-type (GlyT1fl/fl) littermates stained with GlyT1 antiserum. Scale bars, 100 μm (top panels) and 5 μm (bottom panels). (D) Mesodermic expression analysis. Endogenous EGFP fluorescence in coronal spinal cord sections (level L3) of a Hoxb8-Cretg+/RA/EG co-transgenic mouse. Top, overview; bottom, grey matter area. Scale bars, 100 μm. All sections were obtained from 5 – 6 week old mice.
Fig. 4
Fig. 4. β-gal activity in co-transgenic Hoxb8-Cretg+/R26R mice in non-neural tissue
Histological analysis of β–gal activity in 5 - 6 week old co-transgenic Hoxb8-Cretg+/R26R progeny. (A) Skin at the metatarsal region of the hindlimb, (B) kidney, (C) liver, and (D) heart. All sections were from 5 – 6 week old mice, incubated with X-Gal, and counterstained with acidified hematoxylin. Scale bars, 100 μm.
Fig. 5
Fig. 5. Responses to noxious thermal and mechanical stimulation in Hoxb8-Cre mice
Hoxb8-Cre and wild-type littermates (wt) showed virtually identical mechanical thresholds (A) and paw withdrawal latencies upon exposure to noxious heat (B). Mean ± sem (n = 4 - 6 mice / group).

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