Conditional mutagenesis of the Sox2 transcription factor-encoding gene in the mouse: cell type-specific, stage-specific ablation of Sox2 in the nervous system The possibility to mutate specific resident genes of the mouse by gene targeting has revolutionized the study of gene function in higher organisms. On the other hand, homozygous targeted nullā€¯ mutations are often early embryonic lethal, which prevents the study of the genes possible roles at later stages in development. Further, a gene can have distinct roles in different cell types, making the outcome of a constitutiveā€¯ mutation (affecting all cells of the animal, since the beginning of development) often complex to interpret. Finally, when a mutant genotype is viable, but shows pathology in the adult, it is often difficult to discriminate if the gene has a role specifically after birth, or if the defect is a consequence of abnormal development. A recent major advancement to solve these problems has been the development of conditional mutagenesis. Here, gene targeting is used to produce a mutant allele in which the gene is functionally intact, but flanked by lox sites, the substrate of Cre recombinase. Cre, expressed by appropriate transgenes under the control of cell type-specific promoters, will excise the gene in a cell type-specific, stage-specific way. Furher, the use of an inducible form of Cre (CreERT2), which becomes active only following administration of a hormone (tamoxifen), can allow to delete the gene in a temporally regulated way.
The gene encoding Sox2, a conserved transcription factor, is expressed in the totipotent stem cells of the blastocyst inner cell mass, and its absence causes early embryonic lethality due to loss of these cells. At later stages, Sox2 is also expressed in stem cells and undifferentiated precursors throughout the developing nervous system; expression is maintained in the few neural stem cells that persist in the adult brain, as well as in selected neurons. The effects of a knockdownā€¯ mutation, previously generated by our lab by deletion of a neural enhancer, has shown that Sox2 is important also for this later (neural) stem cell type, as well as for neurons of the adult brain. To investigate the effects of complete Sox2 ablation specifically in selected neural cell types (stem cells, or differentiated neurons), and at specific developmental stages (embryonic nervous system, or adult), we have generated a mouse carrying a conditional Sox2 mutation (Sox2 flox).
The project will involve breeding of mice carrying the Sox2 flox mutation to mouse lines expressing different Cre transgenes: initially, we will use nestin-Cre, active specifically in neural stem/ precursor cells since early neural tube development, and the inducible CreERT reconbinase, driven by the ubiquitous beta-actin promoter. We will investigate consequences of Sox2 deletion in vivo and in vitro (the student will chose one as preferential). In vivo, we will analyze embryos at different stages of nervous system development, in which Sox2 will be deleted since early neural tube stages (by nestin-Cre), or at later stages (by CreERT), following tamoxifen treatment of pregnant mothers. We will study the proliferation of neuroepithelial stem/precursor cells and their differentiation into neurons and glia, by BrdU labelling, immunohistochemistry, double fluorescence immunohistochemistry and in situ hybridization. In vitro, we will derive neural stem cell cultures from mutant mice carrying Sox2 flox and the CreERT transgene at various developmental stages (embryo; adult), and we will delete Sox2 by tamoxifen treatment, at specific stages of proliferation or differentiation, to seek effects on stem cells self-renewal, and/or their ability to produce neuronal and glial progeny. These will be evaluated by clonogenic assays as well as immunocytochemistry experiments. |
