Tools for Cre -mediated conditional deletion of floxed alleles from developing cerebellar Purkinje cells.

UNLABELLED: 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 transgenic Cre lines, along with a viral approach, for targeting cerebellar Purkinje cells. Using a combination of a fluorescent reporter line ( Ai14 ) to indicate Cre -mediated recombination and a floxed Dystroglycan line ( Dag1 flox ) we show that reporter expression does not always align precisely with loss of protein. The commonly used Pcp2 Cre line exhibits a gradual mosaic pattern of Cre recombination in Purkinje cells from P7-P14, while loss of Dag1 protein is not complete until P30. Ptf1a Cre drives recombination in precursor cells that give rise to GABAergic neurons in the embryonic cerebellum, including Purkinje cells and molecular layer interneurons. However, due to its transient expression in precursors, Ptf1a Cre results in stochastic loss of Dag1 protein in these neurons. Nestin Cre , which is often described as a "pan-neuronal" Cre line for the central nervous system, does not drive Cre -mediated recombination in Purkinje cells. We identify a Calb1 Cre line that drives efficient and complete recombination in embryonic Purkinje cells, resulting in loss of Dag1 protein before the period of synaptogenesis. AAV8 -mediated delivery of Cre at P0 results in gradual transduction of Purkinje cells 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 Purkinje cells at different developmental stages and illustrate the importance of validating the loss of protein following recombination.

SIGNIFICANCE STATEMENT: The development of Cre lines for targeting gene deletions to defined cellular populations has led to important discoveries in neuroscience. As with any tool, there are inherent limitations that must be carefully considered. Here we describe several Cre lines available for targeting of cerebellar Purkinje cells at various developmental stages. We use the combination of a Cre -dependent fluorescent reporter line and conditional deletion of the synaptic scaffolding molecule Dystroglycan as an example to highlight the potential disconnect between the presence of a fluorescent reporter and the loss of protein.