Cerebellar granule cell-specific and inducible expression of Cre recombinase in the mouse - PubMed (original) (raw)

Cerebellar granule cell-specific and inducible expression of Cre recombinase in the mouse

M Tsujita et al. J Neurosci. 1999.

Abstract

To develop a cell type-specific and temporal regulation system of gene targeting in the cerebellum, we used the NMDA-type glutamate receptor GluRepsilon3 subunit gene and Cre recombinase-progesterone receptor fusion (CrePR) gene in combination. Injection of the CrePR gene placed under the control of the 10 kb 5' region of the GluRepsilon3 gene into C57BL/6 eggs yielded the ECP25 line that strongly expressed the CrePR mRNA selectively in the granule cells of the cerebellum. Using a transgenic mouse carrying a reporter gene for Cre-mediated recombination, we showed that antiprogestins could induce the recombinase activity of CrePR protein in the cerebellar granule cells of the ECP25 line. Thus, the established mouse line will provide a valuable tool to investigate the mechanism of cerebellar function by manipulating molecules in the temporally regulated and granule cell-specific manner.

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Figures

Fig. 1.

Fig. 1.

Inducible activation of CrePR by antiprogestins.A, Structure of CrePR. CrePR protein consists of the NLS of SV40 T antigen, Cre recombinase, and the ligand-binding domain of the human progesterone receptor lacking the C terminal 42 amino acids. The hatched and cross-hatched regions represent the ligand binding and DNA binding domains of the progesterone receptor, respectively. B, Induction of Cre recombinase activity by Org 31806 in NIH3T3 cells. Cell lines transformed with CrePR gene and pNloxZ reporter gene were stained for β-galactosidase activity after incubation with or without 1 μ

m

Org 31806 for 48 hr. Scale bar, 200 μm.

Fig. 2.

Fig. 2.

Isolation and characterization of the ECP25 transgenic mouse line carrying the CrePR gene under the control of the GluRε3 gene promoter. A, Structure of cerebellar granule cell-specific expression vector of CrePR gene. The structure of GluRε3 cDNA and GluRε3 gene are schematically shown_above_ the expression vector. Coding sequence of GluRε3 cDNA is shown by a box. Putative transmembrane or channel-forming segments M1–M4 are indicated. Exons of the GluRε3 gene are shown by filled boxes. The ECP expression vector linearized by _Not_I consists of the 10 kb 5′ region of the GluRε3 gene, the 1.9 kb CrePR gene (hatched box), and the 0.6 kb hG-CSF polyadenylation signal sequence (pA). Cross-hatched bars indicate the probes used for hybridization analyses. E,_Eco_RI; N, Not_I.B, Expression of CrePR mRNA in the cerebellum of the ECP25 line. Total RNA (10 μg) prepared from the forebrain (Fb) and cerebellum (Cb) of ECP25 mouse at P28 was electrophoresed and hybridized with probe A. The_arrowhead indicates the CrePR mRNA. C, Integration of the CrePR gene in the genome of the ECP25 line. Genomic DNA (2 μg each) from the wild-type (WT) and ECP25 mice was digested with Eco_RI, electrophoresed, and hybridized with probes A and B. The intact ECP vector (12.5 kb) is indicated by the filled arrowhead, and endogenous GluRε3 gene fragment hybridized with probe B (11.5 kb) by the_open arrowhead. The >20 kb fragment hybridized with both probes and the 6.6 kb fragment hybridized with probe A represent incomplete copies of the ECP vector.

Fig. 3.

Fig. 3.

Cerebellar granule cell-specific expression of CrePR mRNA in the ECP25 line. A, Negative images of x-ray film autoradiograms showing CrePR mRNA (top) and GluRε3 mRNA (bottom) in parasagittal brain sections. Scale bar, 1 mm. Arrows indicate the border of CrePR mRNA expression in the granular layer of lobule IX. Cb, Cerebellum; Th, thalamus; OB, olfactory bulb. B, Bright-field micrograph at a higher magnification showing the localization of hybridization signals of CrePR mRNA in cerebellar granule cells. Sections were counterstained with methyl green– pyronin. Scale bar, 10 μm.Asterisks indicate the cell body of Purkinje cells.Gr, Granule cell layer; Mol, molecular layer; Pur, Purkinje cell layer.

Fig. 4.

Fig. 4.

Inducible expression of functional Cre recombinase selectively in cerebellar granule cells of the ECP25 line.A, Schema of Cre recombinase assay in vivo. Synthetic steroid Org 31806 activates the recombinase of CrePR protein in the granule cells of the cerebellum. The activated CrePR mediates recombination between two loxP target sites flanking the CAT gene of the reporter, leading to the expression of β-galactosidase gene (lacZ) by CAG promoter (Pcag). B, Staining for β-galactosidase of a parasagittal brain section from a CAG-CAT-Z11 mouse crossed with a mouse expressing Cre recombinase early in the development (K. Nakamura, H. Mori, and M. Mishina, unpublished observations). Scale bar, 1 mm. C, Localization of β-galactosidase expression in the granular layer of the cerebellum of an antiprogestin-injected mouse with the CrePR and CAG-CAT-Z genes. Parasagittal brain sections from the transgenic mice injected with Org 31806 for 4 consecutive days were stained for β-galactosidase 3 d after injection. Scale bar, 1 mm. D, Staining for β-galactosidase of a parasagittal brain section from a control uninjected mouse with the CrePR and CAG-CAT-Z genes. Scale bar, 1 mm.E, High-power micrograph showing the selective expression of β-galactosidase in the cerebellar granule cells of an antiprogestin-injected mouse with the CrePR and CAG-CAT-Z genes. Scale bar, 10 μm. Asterisks indicate the cell body of Purkinje cells. Gr, Granule cell layer;Pur, Purkinje cell layer; Mol, molecular layer.

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