The Cre/loxP recombination system is one of the conditional chromosomal mimics to investigate systemic function of targeted genes, and has been adopted to examine a function of specific gene. For in vivo experiments, rat offers potential advantages of larger body size and progressed ability to accomplish more complex behavioral task as compared to mouse. Here we evaluated a conditional reporter rat line which has red fluorescent protein (tdTomato) gene in the downstream of loxP-flanked STOP cassette.
Firstly we injected AAV-Cre into striatum, hippocampus and cerebellum of the reporter rats to test the conditional expression of tdTomato. Each Cre-immuno-positive cells sparsely located in the injection part and merged with tdTomato. Secondly, site-specific expression was evaluated by in utero electroporation of AcGFP-NCre plasmid. Cortical layer 2/3 neurons were visualized by tdTomato fluorescence in the newborn pup. Thirdly, we evaluated this reporter system using phox2b-Cre driver rat, which specifically expresses Cre in cells of several hindbrain regions involved in the autonomic nervous system including neurons that are responsible for respiratory rhythm generation. When the double transgenic rats with phox2b-Cre and floxed tdTomato were examined at embryonic day (E)12.5, tdTomato was expressed in the neurons of parafacial respiratory group (pFRG), epibranchial ganglia, the forming oculomotor/trochlear nuclei and autonomic ganglia.
It is suggested that the tdTomato was strongly expressed in neurons including Cre with high specificity. Our reporter rat would facilitate the neurophysiological studies and the connectomics of identified neurons which express Cre under a certain promoter.
All animal procedures were conducted in accordance with the guiding principles of Physiological Society of Japan and NIH.