RESUMO
Protein knockdown using the auxin-inducible degron (AID) technology is useful to study protein function in living cells because it induces rapid depletion, which makes it possible to observe an immediate phenotype. However, the current AID system has two major drawbacks: leaky degradation and the requirement for a high dose of auxin. These negative features make it difficult to control precisely the expression level of a protein of interest in living cells and to apply this method to mice. Here, we overcome these problems by taking advantage of a bump-and-hole approach to establish the AID version 2 (AID2) system. AID2, which employs an OsTIR1(F74G) mutant and a ligand, 5-Ph-IAA, shows no detectable leaky degradation, requires a 670-times lower ligand concentration, and achieves even quicker degradation than the conventional AID. We demonstrate successful generation of human cell mutants for genes that were previously difficult to deal with, and show that AID2 achieves rapid target depletion not only in yeast and mammalian cells, but also in mice.
Assuntos
Proteólise/efeitos dos fármacos , Proteômica/métodos , Proteínas Recombinantes de Fusão/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Células HCT116 , Hipocampo/citologia , Humanos , Ácidos Indolacéticos/farmacologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas de Manutenção de Minicromossomo/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , Mutação , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oryza/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Genetic manipulation of protein levels is a promising approach to identify the function of a specific protein in living organisms. Previous studies demonstrated that the auxin-inducible degron strategy provides rapid and reversible degradation of various proteins in fungi and mammalian mitotic cells. In this study, we employed this technology to postmitotic neurons to address whether the auxin-inducible degron system could be applied to the nervous system. Using adeno-associated viruses, we simultaneously introduced enhanced green fluorescent protein (EGFP) fused with an auxin-inducible degron tag and an F-box family protein, TIR1 from Oryza sativa (OsTIR1), into hippocampal neurons from mice. In dissociated hippocampal neurons, EGFP enhanced green fluorescent protein fluorescence signals rapidly decreased when adding a plant hormone, auxin. Furthermore, auxin-induced enhanced green fluorescent protein degradation was also observed in hippocampal acute slices. Taken together, these results open the door for neuroscientists to manipulate protein expression levels by the auxin-inducible degron system in a temporally controlled manner.