RESUMEN
BACKGROUND & AIMS: Human sporadic colorectal cancer (CRC) results from a multistep pathway with sequential acquisition of specific genetic mutations in the colorectal epithelium. Modeling CRC in vivo is critical for understanding the tumor microenvironment. To accurately recapitulate human CRC pathogenesis, mouse models must include these multi-step genetic abnormalities. AIMS: Generate a sporadic CRC model that more closely mimics this multi-step process and use this model to study the role of a novel Let7 target PLAGL2 in CRC pathogenesis. METHODS: We generated a CRISPR/Cas9 somatic mutagenesis mouse model that is inducible and multiplexed for simultaneous inactivation of multiple genes involved in CRC pathogenesis. We used both a doxycycline-inducible transcriptional activator and a dox-inactivated transcriptional repressor to achieve tight, non-leaky expression of the Cas9 nickase. This mouse has transgenic expression of multiple guide RNAs to induce sporadic inactivation in the gut epithelium of four tumor suppressor genes commonly mutated in CRC, Apc, Pten, Smad4 and Trp53. These were crossed to Vil-LCL-PLAGL2 mice which have Cre-inducible overexpression of PLAGL2 in the gut epithelium. RESULTS: These mice exhibited random somatic mutations in all four targeted tumor suppressor genes, resulting in multiple adenomas and adenocarcinomas in the small bowel and colon. Crosses with Vil-LCL-PLAGL2 mice demonstrated that gut-specific PLAGL2 overexpression increased colon tumor growth. CONCLUSIONS: This conditional model represents a new CRISPR/Cas9-mediated mouse model of colorectal carcinogenesis. These mice can be used to investigate the role of novel, previously uncharacterized genes in CRC, in the context of multiple commonly mutated tumor suppressor genes and thus more closely mimic human CRC pathogenesis.
RESUMEN
Diabetes involves the death or dysfunction of pancreatic ß-cells. Analysis of bulk sequencing from human samples and studies using in vitro and in vivo models suggest that endoplasmic reticulum and inflammatory signaling play an important role in diabetes progression. To better characterize cell type-specific stress response, we perform multiplexed single-cell RNA sequencing to define the transcriptional signature of primary human islet cells exposed to endoplasmic reticulum and inflammatory stress. Through comprehensive pair-wise analysis of stress responses across pancreatic endocrine and exocrine cell types, we define changes in gene expression for each cell type under different diabetes-associated stressors. We find that ß-, α-, and ductal cells have the greatest transcriptional response. We utilize stem cell-derived islets to study islet health through the candidate gene CIB1, which was upregulated under stress in primary human islets. Our findings provide insights into cell type-specific responses to diabetes-associated stress and establish a resource to identify targets for diabetes therapeutics.
Asunto(s)
Estrés del Retículo Endoplásmico , Células Secretoras de Insulina , Islotes Pancreáticos , Humanos , Estrés del Retículo Endoplásmico/genética , Islotes Pancreáticos/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Análisis de la Célula Individual , Células Secretoras de Glucagón/metabolismo , Análisis de Secuencia de ARN , Transcriptoma , Estrés FisiológicoRESUMEN
In the original publication [...].