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Tamoxifen-inducible glia-specific Cre mice for somatic mutagenesis in oligodendrocytes and Schwann cells

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Abstract

Inducible transgenesis provides a valuable technique for the analysis of gene function in vivo. We report the generation and characterization of mouse lines carrying glia lineage-specific transgenes expressing an improved variant of the tamoxifen-inducible Cre recombinase, CreERT2, where the recombinase is fused to a mutated ligand binding domain of the human estrogen receptor. Using a PLP-CreERT2 transgene, we have generated mice that show specific inducible Cre function, as analyzed by cross-breeding experiments into the Rosa26 Cre-LacZ reporter line, in developing and adult Schwann cells, in mature myelinating oligodendrocytes, and in undifferentiated NG2-positive oligodendrocyte precursors in the adult. Using a P0Cx-CreERT2 transgene, we have also established mouse lines with inducible Cre function specifically in the Schwann cell lineage. These tamoxifen-inducible CreERT2 lines will allow detailed spatiotemporally controlled analysis of gene functions in loxP-based conditional mutant mice in both developing and adult Schwann cells and in the oligodendrocyte lineage.

Introduction

The generation of conditional mouse mutants is a powerful tool to analyze the function of genes and proteins in their correct physiological context (for review, see Mansuy and Suter, 2000). The technique using the bacteriophage P1 Cre recombinase that removes DNA segments between loxP sites to generate somatic mutations in a controlled way has proven to be exceptionally useful in this regard Lewandoski 2001, Nagy 2000. Further development of the system has yielded the exciting possibility of spatial and temporal control of the recombination event. This was achieved by fusing the mutated ligand-binding domain of either the progesterone receptor (Kellendonk et al., 1999) or the estrogen receptor to the Cre recombinase Danielian et al 1993, Feil et al 1996, Littlewood et al 1995, Metzger et al 1995, Schwenk et al 1998. In the latter, the mutation prevents binding to the natural ligand beta estradiol but allows activation of the recombinase upon association with the synthetic ligand 4-hydroxy (OH)-tamoxifen. The system has been refined to optimize its performance and was successfully used in cultured cells and in developing and adult mice Brocard et al 1997, Danielian et al 1998, Feil et al 1997, Hayashi and McMahon 2002, Imai et al 2001a, Imai et al 2001b, Indra et al 1999, Metzger and Chambon 2001, Vallier et al 2001, Vasioukhin et al 1999, Vooijs et al 2001, Weber et al 2001.

The availability of these novel tools opens the possibility to generate somatic mutations in an extremely versatile way. In the generation of standard null alleles via embryonic stem (ES) cells and overexpression of wild-type or mutant proteins in the mouse, this system overcomes problems associated with early embryonic lethality and allows studying specific gene functions in adult homeostasis without the influence of developmental aberrations. This is particularly important in dissecting disease mechanisms of late-onset degenerative disorders, regeneration processes, and adult nervous system plasticity in mouse models.

In this study, we have used the CreERT2 version of the recombinase that contains a G400V/M543A/L544A triple mutation of the human estrogen receptor and is highly sensitive to nanomolar concentrations of 4-OH-tamoxifen in vitro (Feil et al., 1997). By expressing CreERT2 under appropriate regulatory control, we have generated novel mouse lines that allow temporally and spatially controlled recombination of DNA segments flanked by loxP sites in either Schwann cells and oligodendrocytes together or in Schwann cells alone. These cells are the myelinating glia of the nervous system that are crucially involved in regulation processes during development, repair, and regeneration as well as in devastating demyelinating diseases including multiple sclerosis and peripheral neuropathies. Thus, we anticipate that these mouse lines will be valuable tools to dissect the function of myelinating glia in health and disease.

Section snippets

Generation of transgenic mice expressing Cre-ERT2 in oligodendrocytes and Schwann cells

We generated transgenic mice expressing an improved tamoxifen (TM)-inducible variant of the Cre recombinase (CreERT2) Feil et al 1997, Metzger and Chambon 2001 under the control of two different promoters that are specifically active in oligodendrocytes and/or Schwann cells. To produce the first set of mouse lines, we inserted the CreERT2 open reading frame into an established proteolipid protein (PLP) expression cassette. This construct contains approximately 15 kb of the plp gene regulatory

Discussion

In this study, we describe two novel specific tools for conditional somatic mutagenesis in the mouse. We have combined lineage-specific regulatory elements directing gene expression in oligodendrocytes and Schwann cells with the temporal regulation provided by the TM-inducible CreERT2. As expected, the established PLP gene cassette led to accurate expression of the transgene in oligodendrocytes and Schwann cells. The P0 promoter, supplemented by regulatory regions derived from the connexin32

PLP-CreERT2

A 2.2-kb EcoRI/SalI fragment of the pGS-CreERT2 vector (Feil et al., 1997) containing the CreERT2 cDNA was subcloned into a SmaI cut pBluescript KS+ vector (pBS-ERT2). An EcoRV/SacII fragment containing the Cre cDNA was then cloned into the PLP-SV40 vector (Fuss et al., 2001) that had been linearized with AscI, blunt-ended with Klenow, and then cut with SacII. This leads to an expression cassette containing approximately 15 kb of the mouse plp gene including exons 1 and 2, the CreERT2 cDNA

Acknowledgements

We thank Drs. Ph. Soriano for ROSA26R mice and S. Scherer for the P0C×32 construct and Drs. Ned Mantei, Lukas Sommer, Verdon Taylor, and Philipp Weber for many fruitful discussions and suggestions. This study was supported by the Swiss National Science Foundation, the Swiss Muscle Disease Foundation, the National Center of Competence in Research “Neural Plasticity and Repair,” and the Swiss Bundesamt for Science related to the Commission of the European Communities, specific RTD programme

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