Abstract
ABSTRACTTargeted cell- or region-specific gene recombination is widely used in the functional analysis of genes implicated in development and disease. In the brain, targeted gene recombination has become a mainstream approach to study neurodegeneration or tumorigenesis. The use of the Cre-loxP system to study tumorigenesis in the adult central nervous system (CNS) can be limited, when the promoter (such as GFAP) is also transiently expressed during development, which can result in the recombination of progenies of different lineages. Engineering of transgenic mice expressing Cre recombinase fused to a mutant of the human oestrogen receptor (ER) allows the circumvention of transient developmental Cre expression by inducing recombination in the adult organism. The recombination of loxP sequences occurs only in the presence of tamoxifen. Systemic administration of tamoxifen can, however, exhibit toxicity and might also recombine unwanted cell populations if the promoter driving Cre expression is active at the time of tamoxifen administration. Here, we report that a single site-specific injection of an active derivative of tamoxifen successfully activates Cre recombinase and selectively recombines tumour suppressor genes in neural progenitor cells of the subventricular zone in mice, and we demonstrate its application in a model for the generation of intrinsic brain tumours.
Highlights
The adult central nervous system (CNS) contains several stem cell compartments, which have been studied extensively
In analogy to the more established transepidermal topical application of tamoxifen for Cre-mediated gene recombination, we set out to establish (i) whether injection of active metabolites into the cerebral ventricles can directly activate Cre expression in stem cells, targeting a regionally and spatially defined, specific population of cells and (ii) whether the regional expression in a selected cell population leads to the generation of tumours and how they might compare to tumours induced by recombination due to Cre-expressing adenovirus (Adeno-Cre) or Adeno-glial fibrillary acidic protein (GFAP)-Cre (Henriquez et al, 2013; Jacques et al, 2010)
Optimisation of tamoxifen dosage for intracerebral injection To establish an optimal concentration of 4-OH-TAM or endoxifen to elicit Cre expression with minimal toxicity, we sought to establish the following parameters: (i) optimal concentration for gene recombination, (ii) minimal toxicity and (iii) the optimal volume and carrier (DMSO or ethanol)
Summary
The adult central nervous system (CNS) contains several stem cell compartments, which have been studied extensively. In analogy to the more established transepidermal topical application of tamoxifen for Cre-mediated gene recombination, we set out to establish (i) whether injection of active metabolites into the cerebral ventricles can directly activate Cre expression in stem cells, targeting a regionally and spatially defined, specific population of cells and (ii) whether the regional expression in a selected cell population leads to the generation of tumours and how they might compare to tumours induced by recombination due to Cre-expressing adenovirus (Adeno-Cre) or Adeno-GFAP-Cre (Henriquez et al, 2013; Jacques et al, 2010) To this end, we used mice expressing CreERT2 under control of the endogenous glutamate-aspartate transporter (GLAST) promoter
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