RESUMO
The use of light to control the expression of genes and the activity of proteins is a rapidly expanding field. Whereas many of these approaches use fusion between a light-activable protein and the protein of interest to control the activity of the latter, it is also possible to control the activity of a protein by uncaging a specific ligand. In that context, controlling the activation of a protein fused to the modified estrogen receptor (ERT) by uncaging its ligand cyclofen-OH has emerged as a generic and versatile method to control the activation of proteins quantitatively, quickly, and locally in a live organism. We present that approach and its uses in a variety of physiological contexts.
Assuntos
Optogenética/métodos , Compostos Policíclicos/metabolismo , Receptores de Estrogênio/genética , Animais , Regulação da Expressão Gênica/efeitos da radiação , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Ligantes , Compostos Policíclicos/química , Receptores de Estrogênio/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The use of light to control the expression of genes and the activity of proteins is a rapidly expanding field. While many of these approaches use a fusion between a light activatable protein and the protein of interest to control the activity of the latter, it is also possible to control the activity of a protein by uncaging a specific ligand. In that context, controlling the activation of a protein fused to the modified estrogen receptor (ERT) by uncaging its ligand cyclofen-OH has emerged as a generic and versatile method to control the activation of proteins quantitatively, quickly and locally in a live organism. Here, we present the experimental details behind this approach.
Assuntos
Optogenética/métodos , Compostos Policíclicos/química , Receptores de Estrogênio/genética , Ativação Transcricional , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Expressão Gênica , Luz , Processos Fotoquímicos , Receptores de Estrogênio/química , Peixe-Zebra/embriologiaRESUMO
The zebrafish has become an increasingly popular and valuable cancer model over the past few decades. While most zebrafish cancer models are generated by expressing mammalian oncogenes under tissue-specific promoters, here we describe a method that allows for the precise optical control of oncogene expression in live zebrafish. We utilize this technique to transiently or constitutively activate a typical human oncogene, kRASG12V, in zebrafish embryos and investigate the developmental and tumorigenic phenotypes. We demonstrate the spatiotemporal control of oncogene expression in live zebrafish, and characterize the different tumorigenic probabilities when kRASG12V is expressed transiently or constitutively at different developmental stages. Moreover, we show that light can be used to activate oncogene expression in selected tissues and single cells without tissue-specific promoters. Our work presents a novel approach to initiate and study cancer in zebrafish, and the high spatiotemporal resolution of this method makes it a valuable tool for studying cancer initiation from single cells.