Generating mouse models of degenerative diseases using Cre/lox-mediated in vivo mosaic cell ablation
Masato Fujioka, Hisashi Tokano, Keiko Shiina Fujioka, Hideyuki Okano, Albert S.B. Edge
Masato Fujioka, Hisashi Tokano, Keiko Shiina Fujioka, Hideyuki Okano, Albert S.B. Edge
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Generating mouse models of degenerative diseases using Cre/lox-mediated in vivo mosaic cell ablation

Abstract

Most degenerative diseases begin with a gradual loss of specific cell types before reaching a threshold for symptomatic onset. However, the endogenous regenerative capacities of different tissues are difficult to study, because of the limitations of models for early stages of cell loss. Therefore, we generated a transgenic mouse line (Mos-iCsp3) in which a lox-mismatched Cre/lox cassette can be activated to produce a drug-regulated dimerizable caspase-3. Tissue-restricted Cre expression yielded stochastic Casp3 expression, randomly ablating a subset of specific cell types in a defined domain. The limited and mosaic cell loss led to distinct responses in 3 different tissues targeted using respective Cre mice: reversible, impaired glucose tolerance with normoglycemia in pancreatic β cells; wound healing and irreversible hair loss in the skin; and permanent moderate deafness due to the loss of auditory hair cells in the inner ear. These mice will be important for assessing the repair capacities of tissues and the potential effectiveness of new regenerative therapies.

Authors

Masato Fujioka, Hisashi Tokano, Keiko Shiina Fujioka, Hideyuki Okano, Albert S.B. Edge

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Figure 3

In vitro and in vivo ablation of auditory hair cells in Pouf4f3-Cre;Mos-iCsp3 double transgenics.

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In vitro and in vivo ablation of auditory hair cells in Pouf4f3-Cre;Mos-...
(A and B) Experimental design. (C–G) In vitro ablation of hair cells in organ of Corti explant culture (n = 4 in each group). (C and D) Nuclei of hair cells were visualized by EGFP. (D and E) A significant decrease in hair cells was observed only in double transgenics treated with AP20187 and was apparent at 12 hours. (F) At 72 hours, GFP-positive hair cells were decreased to 49.5% ± 1.6% in double transgenics. (G) The number of supporting cells was unaffected. (H–M) Functional changes in the auditory system were measured (25) by (H and J) DPOAE and (I and K–M) ABR. Amplitude, peak I latency (I, horizontal line), and threshold (I, arrowheads) of the ABR are shown at 16 kHz: significant but moderate deafness was induced only in double transgenics after AP20187 administration (n = 9 and 7 in control and double-transgenic groups, respectively). (N–P) Histological changes in the cochlea (n = 3 and 6 in control and double-transgenic groups, respectively). (N) Mosaic-patterned partial hair cell loss (white arrows) was observed in double transgenics treated with AP20187, confirmed by Pv3 immunostaining and loss of nuclei. The bottom right panel in N was composed from 2 images. (O) Quantification showed 29.3% ± 3.2% of inner hair cells (IHCs) and 23.2% ± 2.6% of outer hair cells (OC1–OC3) were lost, (P) while the density of spiral ganglion neurons was not changed. All data were collected in the 16-kHz area before or 7 days after AP20187 administration. *P < 0.05, **P < 0.01. Scale bar: 50 μm (C); 20 μm (D and N).