Tools forCre-Mediated Conditional Deletion of Floxed Alleles from Developing Cerebellar Purkinje Cells

Abstract

The Cre-lox system is an indispensable tool in neuroscience research for targeting gene deletions to specific cellular populations. Here we assess the utility of several transgenicCrelines, along with a viral approach, for targeting cerebellar Purkinje cells (PCs) in mice. Using a combination of a fluorescent reporter line (Ai14) to indicateCre-mediated recombination and a floxed Dystroglycan line (Dag1flox), we show that reporter expression does not always align precisely with loss of protein. The commonly usedPcp2Creline exhibits a gradual mosaic pattern ofCrerecombination in PCs from Postnatal Day 7 (P7) to P14, while loss of Dag1 protein is not complete until P30.Ptf1aCredrives recombination in precursor cells that give rise to GABAergic neurons in the embryonic cerebellum, including PCs and molecular layer interneurons. However, due to its transient expression in precursors,Ptf1aCreresults in stochastic loss of Dag1 protein in these neurons.NestinCre, which is often described as a “pan-neuronal”Creline for the central nervous system, does not driveCre-mediated recombination in PCs. We identify aCalb1Creline that drives efficient and complete recombination in embryonic PCs, resulting in loss of Dag1 protein before the period of synaptogenesis.AAV8-mediated delivery ofCreat P0 results in gradual transduction of PCs during the second postnatal week, with loss of Dag1 protein not reaching appreciable levels until P35. These results characterize several tools for targeting conditional deletions in cerebellar PCs at different developmental stages and illustrate the importance of validating the loss of protein following recombination.