Art by Jake Upton
By Alex Hepp
In the last several decades, new techniques for biological applications of genetic editing have arisen, including the CRISPR/Cas9, ZFNs, TALENs, and Cre-loxP systems. These techniques are often combined with cellular labeling techniques to tag cells of interest with colored fluorescent proteins.
A recently introduced gene labeling strategy called Brainbow involves hundreds of different hues that can be generated among fluorescent proteins. The Brainbow technique combines the three primary colors: red, green, and blue to generate all the colors in the visual spectrum and expresses them in differing ratios within each targeted cell. The resulting color combination serves as cellular identification under a microscope. The combinatorial effect of these expressed colors can be used as cellular tags for tracing axons and neuronal cell bodies, as well as following the development of targeted cells, and analyzing cell lineage.
Brainbow is typically used in combination with the Cre-loxP recombination system. Cre-loxP performs a site-specific DNA recombination technique that causes the individual nucleic acids of DNA to be deleted, inserted, or translocated. In this method, the fluorescent proteins are incorporated into a transgene, a gene that is transferred into the genome of another organism, and the Cre-loxP system randomly expresses the color transmitted. This allows for analysis of neural connectivity and progenitor cell migration and lineage in the development of animal models, which can be translated to a deeper understanding of our own genome and cellular development.
More specifically, the Cre-loxP recombination system involves the enzyme Cre-recombinase which is able to recognize two directly repeated loxP sites in the DNA and remove the sequence between them. The Cre-recombinase can be expressed in a cell through a variety of techniques that allows insertion of genes into other organisms. One of these techniques uses a sequence called a promoter, that may only be expressed in certain cell types. Thus, the expression of Cre-recombinase can also be limited to specific cells and when used in combination with Brainbow, cells can be targeted to express a color scheme.
In studies completed using the Brainbow technique on transgenic mice (mice who contain genetic material from a transgenes), scientists were able to label a series of different cellular aspects.
(A) Brainbow 3.0 constructing transgenic mice, process of incorporating antigenically distinct fluorescence proteins in mice for membrane labeling and antibody amplification in the extracellular matrix. (D) Brainbow 3.2 increasing fluorescent levels to display five motor axons expressed in different colors. (E) Cerebellum from a Brainbow 3.1 Cre-mouse. Several Purkinje cells labeled by distinct colors.
Brainbow has been used on a variety of animal models including drosophila, mice, nematodes, zebrafish and some plant types. In figure 2, (C) cellular differentiation in the pectoral fin of a zebrafish. (D) sensory neurons in the ventrolateral body wall of a drosophila larva.
Understanding and interpreting new genetic editing tools are crucial to scientific progression and the future development of other cellular and tissue specific genome editing and pharmaceutical agents. The combination of a variety of genetic techniques such as the Cre-loxP system and Brainbow is only one example of thousands that has spurred scientific exploration of our cells, the development of axons through the nervous system, progress of individual cells during development, and analyzing cell lineage.
Cai, D., Cohen, K. B., Luo, T., Lichtman, J. W., & Sanes, J. R. (2013). Improved tools for the Brainbow toolbox. Nature Methods, 10(6), 540–547. https://doi-org.ezproxy.uvm.edu/10.1038/nmeth.2450
Kim, H., Kim, M., Im, S. K., & Fang, S. (2018). Mouse Cre-LoxP system: general principles to determine tissue-specific roles of target genes. Laboratory Animal Research, 34(4), 147–159. https://doi-org.ezproxy.uvm.edu/10.5625/lar.2018.34.4.147
Weissman, T. A., & Pan, Y. A. (2015). Brainbow: new resources and emerging biological applications for multicolor genetic labeling and analysis. Genetics, 199(2), 293–306. https://doi-org.ezproxy.uvm.edu/10.1534/genetics.114.172510