Abstract
Modeling gliomas in genetically engineered mouse models (GEMMs) is the gold standard for recapitulation of the human disease. However, with its heterogeneity and myriad genetic mutations, this becomes a difficult, if not impossible task. Our lab has recently developed an in vivo method of transgenesis called mosaic analysis by dual recombinase-mediated cassette exchange (MADR) that involves genetic recombination at the Rosa26 locus, allowing for consistent transgenic expression in mutant cells. Using electroporation-mediated delivery, DNA plasmids are injected into the lateral ventricle of the brain, followed by application of an electric current that allows the DNA to be taken up by the shocked cells. MADR donor plasmids are then inserted into this locus using FlpO and Cre recombinases. The MADR donor plasmid is a promoter-less vector with a cistron encoding transgenes (e.g., tumor driver genes and/or reporter genes separated by P2A self-cleaving peptides in order to generate multiple proteins from a single cistron) flanked by loxP and FRT sites. For tertiary recombinase expression, VCre is added (with an additional P2A) to the donor. Further, for inducible control of VCre expression, we have developed three separate plasmids: (1) VCre preceded by destabilized domain (DD), as well as dual flanking domains of (2) estrogen ligand binding domain (ERT2) and (3) progesterone receptor (PR). Finally, a flip excision (FlEx) cistron is co-electroporated which can be induced by VCre. Induction of any number of additional transgenes can thus be included such as genes associated with tumor progression or candidate elements for therapeutic mimicry. Incorporating a tertiary level of inducible recombinase expression through VCre, therefore, substantially widens the utility of this method to not only recapitulate the clinical profile of tumor progression, but also to explore different temporal and spatial cells of origin by regulation of this tertiary recombinase.