RESUMO
Mice engineered for conditional, cell type-specific gene inactivation have dominated the field of mouse genetics because of the high efficiency of Cre-loxP-mediated recombination. Recent advances in CRISPR/Cas9 technologies have provided alternatives for rapid gene mutagenesis for loss-of-function (LOF) analysis. Whether these strategies can be streamlined for rapid genetic analysis with the efficiencies comparable with those of conventional genetic approaches has yet to be established. We show that a single adeno-associated viral (AAV) vector containing a recombinase-dependent Staphylococcus aureus Cas9 (SaCas9) and a single guide RNA (sgRNA) are as efficient as conventional conditional gene knockout and can be adapted for use in either Cre- or Flp-driver mouse lines. The efficacy of this approach is demonstrated for the analysis of GABAergic, glutamatergic, and monoaminergic neurotransmission. Using this strategy, we reveal insight into the role of GABAergic regulation of midbrain GABA-producing neurons in psychomotor activation.
Assuntos
Envelhecimento/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Dependovirus/genética , Vetores Genéticos/metabolismo , Mutagênese/genética , Sistema Nervoso/metabolismo , Animais , Sequência de Bases , Linhagem Celular , DNA Nucleotidiltransferases/metabolismo , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Ácido Glutâmico/metabolismo , Camundongos Endogâmicos C57BL , Mutação/genética , Optogenética , FenótipoRESUMO
The heterotrophic marine bacterium, Ruegeria pomeroyi, was experimentally cultured under environmentally realistic carbon conditions and with a tracer-level addition of 13C-labeled leucine to track bacterial protein biosynthesis through growth phases. A combination of methods allowed observation of real-time bacterial protein production to understand metabolic priorities through the different growth phases. Over 2000 proteins were identified in each experimental culture from exponential and stationary growth phases. Within two hours of the 13C-labeled leucine addition, R. pomeroyi significantly assimilated the newly encountered substrate into new proteins. This dataset provides a fundamental baseline for understanding growth phase differences in molecular physiology of a cosmopolitan marine bacterium.
