DATA ANALYSIS PROBLEM

Livet, J., T.A. Weissman, H.N. Kang, R.W. Draft, J. Lu, R.A. Bennis, J.R. Sanes, and J.W. Lichtman. 2007. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450:56–62.

Question 14.15

Site-specific recombination and transposition are regularly used in biotechnology. An elegant use of site-specific recombination is found in a 2007 report by Livet and colleagues. One of the challenges in brain research is the sheer complexity of the neuronal network. Tracing one neuron to elucidate its connections was nearly impossible until the advent of the “brainbow” technology (see Highlight 14-1). Researchers placed genes for different colored variants of green fluorescent protein (GFP; see Figure 7-22) in cassettes, with the various GFP genes separated by lox sites recognized by the site-specific recombinase Cre (Figure 1a), and inserted the cassettes into the mouse genome. The variants in this example are red fluorescent protein (RFP), yellow fluorescent protein (YFP), and cyan fluorescent protein (CFP). Each cassette was inserted at FRT sites, using a separate site-specific recombinase, Flp. Multiple cassettes were inserted into the genomes of some mice (producing the brainbow in Figure 1b).

The Cre recombinase was inserted into the genome of a different group of mice, the cloned enzyme structured so that it was expressed uniquely in brain tissue, for a brief time, during neuron development. When mice containing the cassettes were mated to mice containing the cloned Cre recombinase, Cre-mediated recombination created a unique pattern of expression of GFP variants in each mature neuron of the heterozygous offspring, effectively coloring the neurons. The vivid results are shown in Highlight 14-1.

  1. Suggest why the Flp recombination system rather than Cre-lox was used to insert the cassettes into the mouse genome.

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    FIGURE 1
  2. If one GFP cassette was already present in the mouse genome, how could more cassettes be added?

  3. In the cassette shown in Figure 1a, two different lox sites are used. What sequences in the lox sites must be different to prevent the two sites from recombining with each other?

  4. When two or more different GFP variants are expressed in a neuron, the final color is a blend that reflects the amount of each GFP present. In mice with three cassettes, one GFP variant is expressed from each cassette (assuming that the lox sites preclude recombination between cassettes). Ten different colors are possible. Describe the combinations leading to the different colors.