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Erzeugung von Maus-Linien für die induzierbare Expansion neuronaler Stammzellen

 

We use in utero electroporation as a system to acutely and tissue-specifically manipulate gene expression of neural stem cells of developing mouse embryos (Calegari et al., 2002). With this system we found that overexpression of cdk4/cyclinD1 in neural progenitors shortens their G1 phase by 30% (6.0 vs. 9.3 hours) while inhibiting their switch to neurogenesis by 70% (Fig 1).

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Legend Figure 2: From Lange et al., in press. A) Dual reporter/cdk4 or cyclinD1 plasmids. B) Representation of in vivo electroporation after injection of DNA (left) and its effect one day after targeting with a GFP construct (right). C) Control GFP/RFP (left) or cdk4/cyclinD1 (right) plasmids were delivered to neural progenitors at E13.5. One day later, proportion of targeted cells (and their progeny) was determined in different cortical areas: ventricular (VZ), sub-ventricular (SVZ) and intermediate (IZ) zone. D) Proportion of neurons in the population of targeted cells.

In addition, under these conditions we observed an increased generation and expansion of basal progenitors that contributed to a 30% thickening of the subventricular zone and, ultimately, to a three-fold increase in cortical surface area containing neurons genereted from them (Fig 2). Finally, RNAi for cdk4/cyclinD1 revealed to induce the opposite effects i.e. a lengthening of the cell cycle, increased neurogenesis, and consumption of basal progenitors. Therefore, we conclude, G1 lengthening is both necessary and sufficient to induce the switch of neural stem cells from proliferation to neurogenesis (lange et al., in press).

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Legend Figure 3: From Lange et al., in press. A) Cell fate analysis of targeted cells (red) immunolabeled for a marker of basal (Tbr2; white) or neurogenic (Tis21; green) progenitors. B) Proliferating basal progenitors (Tbr2+/Tis21–) were scored in the population of cdk4/cyclinD1-targeted (black) or control (white) cells of five brains (symbols). C) Analyses of cortical surface area contributed by targeted progenitors at P0 by 3D reconstruction or the cortex containing RFP+ cells (red). D) quantification as in C.

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Funding program:

DFG