Investigation of Beige Fat Biology and Metabolism Using the CRISPR SunTag-p65-HSF1 Activation System

Autor(es)

Bladimir, Fernando ; de Oliveira, Vanessa ; Henrique, Carlos

• Tipo de documento: Artigo Científico
• Fonte: JOVE-JOURNAL OF VISUALIZED EXPERIMENTS; v. N/A, n. 191, p. 1-pg., 2023-01-01.

Resumo

Clustered regularly interspaced short palindromic repeats (CRISPR) technology has prompted a revolution in biology, and recent tools have been applied far beyond the originally described gene editing. The CRISPR activation (CRISPRa) system combines the catalytically inactive Cas9 (dCas9) protein with distinct transcription modules to induce endogenous gene expression. SunTag-p65-HSF1 (SPH) is a recently developed CRISPRa technology that combines components of synergistic activation mediators (SAMs) with the SunTag activators. This system allows the overexpression of single or multiple genes by designing a customized single-guide RNA (sgRNA). In this study, a previously developed SPH mouse was used to generate a conditional mouse expressing SPH in adipocytes (adiponectin Cre lineage), named AdipoSPH. To induce a white-to-beige fat (browning) phenotype, an adenoassociated virus (AAV) carrying sgRNA targeting the endogenous Prdm16 gene (a well-established transcription factor related to brown and beige fat development) was injected into the inguinal white adipose tissue (iWAT). This mouse model induced the expression of endogenous Prdm16 and activated the thermogenic gene program. Moreover, in vitro SPH-induced Prdm16 overexpression enhanced the oxygen consumption of beige adipocytes, phenocopying the results of a previous Prdm16 transgenic mouse model. Thus, this protocol describes a versatile, cost-effective, and time-effective mouse model for investigating adipose tissue biology. (AU)

• Processo FAPESP: 19/15025-5 - Caracterização do complexo transcricional do PRDM16 envolvido na ação repressora de genes relacionados à síntese de ceramidas em adipócitos
• Beneficiário: Carlos Henrique Grossi Sponton
• Modalidade de apoio: Auxílio à Pesquisa - Jovens Pesquisadores
• Processo FAPESP: 20/14725-0 - Caracterização do complexo transcricional do PRDM16 envolvido na ação repressora de genes relacionados à síntese de ceramidas em adipócitos
• Beneficiário: Fernando Bladimir Valdivieso Rivera
• Modalidade de apoio: Bolsas no Brasil - Doutorado Direto
• Processo FAPESP: 21/11841-2 - Caracterização do complexo transcricional do PRDM16 envolvido na ação repressora de genes relacionados à síntese de ceramidas em adipócitos
• Beneficiário: Vanessa de Oliveira Furino
• Modalidade de apoio: Bolsas no Brasil - Programa Capacitação - Treinamento Técnico
• Processo FAPESP: 20/09308-1 - Caracterização do complexo transcricional do PRDM16 envolvido na ação repressora de genes relacionados à síntese de ceramidas em adipócitos
• Beneficiário: Carlos Henrique Grossi Sponton
• Modalidade de apoio: Bolsas no Brasil - Jovens Pesquisadores