Neural circuit analysis using a novel intersectional split intein-mediated split-Cre recombinase system

Abstract

Keywords: 23Split intein, split-cre recombinase, circuit mapping, projection neurons, retrograde virus, 24 DLX6, GABA, transgenic mouse. 25 Khoo et al. 2 Abstract 26The defining features of a neuron are its functional and anatomical connections with 27 thousands of other neurons in the brain. Together, these neurons form functional networks 28 that direct animal behaviour. Current approaches that allow the interrogation of specific 29 populations of neurons and neural circuits rely heavily on targeting their gene expression 30 profiles or connectivity. However, these approaches are often unable to delineate specific 31 neuronal populations. Here, we developed a novel intersectional split intein-mediated split-32Cre recombinase system that can selectively label specific types of neurons based on their 33 gene expression profiles and structural connectivity. We developed this system by splitting 34Cre recombinase into two fragments with evolved split inteins and subsequently expressed 35 one fragment under the influence of a cell type-specific promoter in a transgenic animal, and 36 delivered the other fragment via retrograde viral gene transfer. This approach results in the 37 reconstitution of Cre recombinase in only specific population of neurons projecting from a 38 specific brain region or in those of a specific neuronal type. Taken together, our split intein-39 based split-Cre system will be useful for sophisticated characterization of mammalian brain 40 circuits. 41 Khoo et al.

Visualizing Cathepsin K‐Cre Expression at the Single‐Cell Level with GFP Reporters

“…Because of the widespread expression of Ctsk-Cre in cells other than osteoclasts, it is essential to develop dual promotermediated Cre systems for more precise genetic targeting to osteoclasts, for example, split-Cre (53)(54)(55)(56) and Dre-Cre. (57)(58)(59) Recent progress in bulk sequencing and scRNA-seq will provide an opportunity to choose the optimal combination of two promoters to allow Cre to be more accurately expressed in osteoclasts for genetic manipulation.…”