These Thy1-Brainbow 2.1 (line R) transgenic mice are viable and fertile. The mice possess two invertible DNA segments (four fluorescent protein sequences in total) uniquely positioned in tandem and flanked with LoxP sites to generate a larger number of recombination outcomes; allowing stochastic expression of multiple fluorescent proteins from a single transgene. Although the fluorescent protein immediately adjacent to the promoter, hrGFPII (with nuclear localization signal), was designed to be expressed prior to Cre-mediated recombination, basal hrGFPII expression may not be observed in mouse tissues. When bred to Cre recombinase expressing mice, however, the resulting offspring can have one of three different inversions for each transgene in each cell of the cre expressing tissue(s). In addition, two excision events may reduce the construct to one of two single invertible DNA segments which can continue to invert as long as cre is present. These different recombination possibilities can be used to express four genes (hrGFPII, mYFP, tdimer2(12) (RFP), or mCerulean (CFP)) to the peripheral and central neurons. A nuclear localization signal targets the hrGFPII to the nucleus, a palmitoylation sequence tethers the mCerulean (CFP) to the membrane (allowing clear labeling of axonal processes), and cytoplasmic tdimer2(12) (RFP) and mYFP better labeled neuronal cell bodies and dendrites. Integration of tandem transgene copies yields combinatorial fluorescent protein expression in each cell, and thus many possible cell colors, providing a way to distinguish adjacent neurons and visualize other cellular interactions. Of note, the single FRT site inserted at the 5' end of the transgene allows tandem transgene copy number reduction through Flp-mediated recombination if desired. These Brainbow 2.1 (founder line R) mice allow labeling of individual neurons in peripheral and central neurons with nuclear localized, membrane-targeted, or cytoplasmic fluorescent proteins in cre recombined cells, and may also be useful in conjunction with other Brainbow strains (Stock No. 007901, Stock No. 007910, Stock No. 007911) for neurobiological studies. The Jackson Laboratory 2024, used with permission.