Cre Adeno-associated virus(AAV Serotype 2)

Despite the advantages of the Cre/loxP recombination system in animal models, its application in rats has lagged behind that in mice due to the lack of relevant reporter strains. Here, researchers generated a floxed STOP tdTomato rat that conditionally expresses a red fluorescent protein variant (tdTomato) in the presence of exogenous Cre recombinase. The tdTomato signal vividly visualized neurons, including their projection fibers and spines, without any histological enhancement. In addition, a transgenic rat line (FLAME) that ubiquitously expresses tdTomato was successfully established by injecting intracytoplasmic Cre mRNA into fertilized eggs. The rat reporter system here will facilitate connectome studies and visualization of the fine structures of genetically identified cells for extended periods of time in vivo and in vitro. In addition, FLAME is an ideal model for organ transplantation studies due to the improved traceability of cells/tissues.

Here, to examine tdTomato expression after complete excision of the STOP cassette filler, AAV2-Cre virus vectors containing Cre was injected into the striatum (n = 8), hippocampus (n = 7), and cerebellum (n = 2) of heterozygous rats obtained by crossing male NBRP-0734 with wild-type female LE rats. In all rats, bright red fluorescence was detected only in the area surrounding the injection site. Cre-immunoreactive nuclei (Alexa Fluor 488) and tdTomato immunoreactivity (Alexa Fluor 546) colocalized within the same cells (Figure 1A). Almost all cells expressing Cre were successfully recombined (Figure 1B). Even after paraformaldehyde fixation, tdTomato fluorescence signals were retained, and fine structures such as spines could still be resolved without immunohistochemical enhancement (Figures 1C and 1D). Upon closer inspection, tdTomato was expressed primarily in neurons, presumably the neuronal target of the virus serotype. The fluorescence from the projection fibers was visible even without histological enhancement.

Figure 1. AAV2-Cre-mediated reporter gene expression in the brain. (Igarashi H, et al., 2016)