Cre recombinase-mediated DNA recombination is an established method for conditional control of gene expression in animal models. Regulation of its activity has been accomplished to impart spatial and/or temporal control over recombination of the target gene. In this chapter, optical control of Cre recombinase in developing zebrafish embryos through genetic code expansion is discussed. This method takes advantage of an evolved aminoacyl tRNA synthetase and tRNA pair that can incorporate an unnatural amino acid (UAA) into proteins in response to an amber stop codon (TAG). Genetic code expansion is used to replace a lysine residue critical to Cre recombinase function with a photocaged analogue of lysine, successfully blocking DNA recombination until irradiation with 405nm light. Use of optically controlled Cre recombinase for cell-lineage tracing experiments in zebrafish embryos is highlighted, demonstrating the ability to target small populations of cells at different developmental time points for recombination. Optically controlled Cre recombinase showed no background activity and precise activation upon irradiation, making it a useful new tool for studying development and disease in the zebrafish embryo.
Keywords: Caged protein; Cre recombinase; DNA recombination; Genetic code expansion; Light-activation; Zebrafish.
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