Efficient conditional and promoter-specific in vivo expression of cDNAs of choice by taking advantage of recombinase-mediated cassette exchange using FlEx gene traps

Recombinase-mediated cassette exchange (RMCE) exploits the possibility to unidirectionally exchange any genetic material flanked by heterotypic recombinase recognition sites (RRS) with target sites in the genome. Due to a limited number of available pre-fabricated target sites, RMCE in mouse embryonic stem (ES) cells has not been tapped to its full potential to date. Here, we introduce a universal system, which allows the targeted insertion of any given transcriptional unit into 85 742 previously annotated retroviral conditional gene trap insertions, representing 7013 independent genes in mouse ES cells, by RMCE. This system can be used to express any given cDNA under the control of endogenous trapped promoters in vivo, as well as for the generation of transposon ‘launch pads’ for chromosomal region-specific ‘Sleeping Beauty’ insertional mutagenesis. Moreover, transcription of the gene-of-interest is only activated upon Cre-recombinase activity, a feature that adds conditionality to this expression system, which is demonstrated in vivo. The use of the RMCE system presented in this work requires one single-cloning step followed by one overnight gateway clonase reaction and subsequent cassette exchange in ES cells with efficiencies of 40% in average

Manipulation des Maus Genoms mit Transposon und Rekombinase Techniken

Random insertional mutagenesis can reveal a lot of insight in specific gene function and phenotypical consequences of the affected gene. The generation of such mutations in the model organism mouse is due to the striking synteny of mouse and human genomes especially interesting with respect to human genetic diseases. In this work, the establishment of a transposon-based mutagenic in vivo screen was the goal. The combination of this method with recombinase techniques even enables the directed mutagenesis of critical regions, to identify new candidate genes.Zufällig generierte insertionelle Genmutationen können viel Aufschluss über spezifische Funktion und phänotypische Auswirkungen des beeinträchtigten Gens liefern. Die Erzeugung solcher Mutationen im Modelorganismus Maus ist besonders im Kontext menschlicher Gendefekte interessant, da eine hohe Syntänie beider Genome besteht. In dieser Arbeit wurde versucht eine Transposon basierte Mutagenese („springende Gene“) in vivo zu etablieren. Zudem wurde diese Methode mit Rekombinase Techniken gekoppelt, um zielgerichtet kritische Regionen im Genom zu manipulieren, und dadurch neue Kandidatengene zu identifizieren

MicroRNA Cluster miR-17-92 Regulates Neural Stem Cell Expansion and Transition to Intermediate Progenitors in the Developing Mouse Neocortex

SummaryDuring development of the embryonic neocortex, tightly regulated expansion of neural stem cells (NSCs) and their transition to intermediate progenitors (IPs) are critical for normal cortical formation and function. Molecular mechanisms that regulate NSC expansion and transition remain unclear. Here, we demonstrate that the microRNA (miRNA) miR-17-92 cluster is required for maintaining proper populations of cortical radial glial cells (RGCs) and IPs through repression of Pten and Tbr2 protein. Knockout of miR-17-92 and its paralogs specifically in the developing neocortex restricts NSC proliferation, suppresses RGC expansion, and promotes transition of RGCs to IPs. Moreover, Pten and Tbr2 protectors specifically block silencing activities of endogenous miR-17-92 and control proper numbers of RGCs and IPs in vivo. Our results demonstrate a critical role for miRNAs in promoting NSC proliferation and modulating the cell-fate decision of generating distinct neural progenitors in the developing neocortex