Figure 3. In vivo barcode generation.

One strategy for generating sufficient diversity to barcode every neuron's DNA uniquely is shown above. In this strategy, inspired by Brainbow [16], each cell's genome contains a cassette consisting of a sequence of short unique barcode elements A…E… (top). Each barcode element is flanked by recombination sites (triangles). Upon expression of a suitable recombinase, these barcode elements shuffle and invert (shown here by inverted letter). The theoretical diversity that can be generated by this is 2^NN!, where N is the number of barcode elements. For a cassette containing N = 12 elements, the theoretical diversity is 2×10¹², far more than needed to barcode the 10⁸ neurons in a mouse brain uniquely. Note that if a conventional recombinase like cre or flp is used here, excision will dominate over inversion and the resulting diversity scales with the number of barcode elements N. To avoid excision we use RCI [24], a recombinase that inverts but does excise.