Abstract
The production of cell-type- and age-specific genetically modified mice is a powerful approach for unraveling unknown gene functions. Here, we present a simple and timesaving method that enables adeno-associated virus (AAV)-mediated cell type- and age-specific recombination in floxed mice. To achieve astrocyte-specific recombination in floxed Ai14 reporter mice, we intravenously injected blood-brain barrier-penetrating AAV-PHP.eB vectors expressing Cre recombinase (Cre) using the astrocyte-specific mouse glial fibrillary acidic protein (mGfaABC1D) promoter. However, we observed non-specific neuron-predominant transduction despite the use of an astrocyte-specific promoter. We speculated that subtle but continuous Cre expression in non-astrocytic cells triggers recombination, and that excess production of Cre in astrocytes inhibits recombination by forming Cre–DNA aggregates. Here, we resolved this paradoxical event by dividing a single AAV into two mGfaABC1D-promoter-driving AAV vectors, one expressing codon-optimized flippase (FlpO) and another expressing flippase recognition target-flanked rapidly degrading Cre (dCre), together with switching the neuron-tropic PHP.eB capsid to astrocyte-tropic AAV-F. Moreover, we found that the FlpO – dCre system with a target cell-tropic capsid can also function in neuron-targeting recombination in floxed mice.
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More From: Molecular Therapy - Methods & Clinical Development
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