Cre-mediated recombination efficiency and transgene expression patterns of three retinal bipolar cell-expressing Cre...

Retinal bipolar cells, comprising multiple types, play an essential role in segregating visual information into multiple parallel pathways in the retina. The ability to manipulate gene expression in specific bipolar cell type(s) in the retina is important for understanding the molecular basis of their normal physiological functions and diseases/disorders. The Cre/LoxP recombination system has become an important tool for allowing gene manipulation in vivo, especially with the increasing availability of cell- and tissue-specific Cre transgenic mouse lines. Detailed in vivo examination of the Cre/LoxP recombination efficiency and the transgene expression patterns for cell- and tissue-specific Cre transgenic mouse lines is essential for evaluating their utility. In this study, we investigated the Cre-mediated recombination efficiency and transgene expression patterns of retinal bipolar cell-expressing Cre transgenic lines by crossing with a Cre reporter mouse line and through Cre-dependent recombinant adeno-associated virus (rAAV) vector-mediated transgene delivery.

Three retinal bipolar cell-expressing Cre-transgenic mouse lines, 5-HTR2a-cre, Pcp2-cre, and Chx10-cre, were crossed with a strong Cre reporter mouse line that expresses a red fluorescent protein variant, tdTomato. rAAV2 vectors carrying a double-floxed inverted open-reading frame sequence encoding channelrhodopsin-2-mCherry (ChR2-mCherry) driven by a ubiquitous neuronal EF1α or a ubiquitous CMV promoter with a rAAV2 capsid mutation (Y444F) were injected into the intravitreal space of the eyes. Immunohistochemistry using retinal bipolar cell type-specific markers was performed to examine Cre-mediated recombination efficiency and the transgene expression patterns in bipolar cells in retinal whole mounts and vertical sections.

For the 5-HTR2a-cre and Pcp2-cre mouse lines, the expression pattern of the Cre-mediated recombination by crossing the reporter line largely resembled the expression pattern of Cre. The bipolar cells showing Cre-mediated recombination in the 5-HTR2a-cre line and the Pcp2-cre line were predominantly type 4 cone bipolar cells and rod bipolar cells, respectively. For the Chx10-cre mouse line, the expression pattern of the Cre-mediated recombination by crossing the reporter line was different from that of Cre. The Cre-mediated transgene expression in retinal bipolar cells in the Chx10-cre line was not observed by crossing with the reporter mouse line but through Cre-dependent rAAV vector delivery. A rAAV2 vector with the combination of a CMV promoter and the Y444F capsid mutation achieved Cre-dependent transgene expression in retinal bipolar cells.

The retinal bipolar cell-expressing Cre-transgenic lines and the Cre-dependent rAAV vector reported in this study could be valuable tools for gene targeting and manipulation in retinal bipolar cells in mice.