Transgenic Mice with Integrated Brainbow 3.0 Cassette Exhibit Non-Uniform Cytoplasmic Distribution of mOrange2 Protein in Neurons

Objective: Brainbow 3.0 is a genetic labeling technique that enables the visualization of individual cells via production unique combinations of fluorescent proteins. This approach utilizes an expression cassette that consists three fluorescent protein genes—mOrange2, eGFP, and mKate2, which are separated by lox sites, recognized by the Cre recombinase. Our goal was to generate a transgenic mouse line carrying the CAG-Brainbow 3.0 cassette and to analyze the intracellular localization of the fluorescent protein mOrange2 in these mice. Methods: A double-stranded DNA fragment containing the CAG promoter and Brainbow 3.0 cassette was microinjected into the pronuclei of fertilized C57BL/6 mouse oocytes. The presence of the transgene insert in the transgenic mice was assessed by PCR genotyping. Transgene expression was evaluated using fluorescence and confocal microscopy. Results and discussion: Transgenic mice were obtained with random genomic integration of the Brainbow 3.0 fluorescent protein cassette under the control of the constitutive CAG promoter. Analysis of transgene expression revealed that the constitutively synthesized mOrange2 protein forms intracellular clusters in neurons and does not exhibit membrane localization. The observed mOrange2 clusters were not toxic to neurons. Conclusions: mOrange2, despite being fused to a farnesylation signal, fails to localize uniformly to the plasma membrane in neurons. While these aggregates do not cause cellular toxicity, they limit the utility of this line for applications requiring accurate membrane tracing. However, the line’s stable expression and strong signal make it suitable for imaging of neurons in vivo. This unexpected behavior of mOrange2 in neurons also makes the line potentially useful for investigating the intracellular trafficking or retention of lipidated proteins.