Brainbow - Methods

Methods

Brainbow techniques rely on the Cre-Lox recombination, in which the protein Cre recombinase drives inversion or excision of DNA between loxP sites. The original Brainbow method includes both Brainbow-1 and Brainbow-2, which utilize different forms of cre/lox recombination. Brainbow-1 uses DNA constructs with different fluorescent protein genes (XFPs) separated by mutant and canonical forms of loxP. This creates a set of mutually exclusive excision possibilities, since cre mediated recombination occurs only between identical loxP sites. After recombination occurs, the fluorescent protein that is left directly after the promoter is uniquely expressed. Thus, a construct with four XFPs separated by three different loxP sites, three excision events, and the original construct can produce four different fluorescent proteins.

Brainbow-2 uses Cre excision and inversion to allow multiple expression possibilities in a given construct. In one DNA segment with two oppositely oriented XFPs, Cre will induce a random inversion event that leaves one fluorescent protein in the proper orientation for expression. If two of these invertible sequences are aligned, three different inversion events are possible. When excision events are also considered, one of four fluorescent proteins will be expressed for a given combination of Cre excisions and inversions. For both Brainbow-1 and-2, the expression of a given XFP is a stochastic, or random, event. Brainbow is implemented in vivo by crossing two transgenic organism strains: one that expresses the Cre protein and another that has been transfected with several versions of a loxP/XFP construct. Using multiple copies of the transgene allows the XFPs to combine in a way that can give one of approximately 100 different colors. Thus, each neuron is labeled with a different hue based on its given combinatorial and stochastic expression of fluorescent proteins. In order to elucidate differential XFP expression patterns into a visible form, brain slices are imaged with confocal microscopy. When exposed to a photon with its particular excitation wavelength, each fluorophore emits a signal that is collected into a red, green, or blue channel, and the resultant light combination is analyzed with data analysis software. Superimposition of differentially colored neurons allows visual disentanglement of complicated neural circuits.

Brainbow has predominantly been tested in mice to date; however, the basic technique described above has also been modified for use in more recent studies since the advent of the original method introduced in 2007.