Virus-mediated gene knockdown in intact pancreatic islets is technically challenging due to poor infection of the center of the islet. Because the cells that do not have knockdown have normal insulin secretion, measuring changes in insulin secretion after gene knockdown is challenging. We describe a method to monitor insulin secretion from only the beta cells with knockdown of a gene of interest in intact islets using a single lentivirus containing a guide RNA, a luciferase insulin secretion reporter and a dCas9-KRAB cassette. This method allows rapid and inexpensive monitoring of insulin secretion from only those beta cells with knockdown, circumventing the problem of incomplete islet infection.

由于胰岛中心感染率低，病毒介导的完整胰岛基因敲除在技术上具有挑战性。因为没有被击倒的细胞具有正常的胰岛素分泌，所以测量基因击倒后胰岛素分泌的变化是具有挑战性的。我们描述了一种方法，使用含有引导 RNA、萤光素酶胰岛素分泌报告基因和 dCas9-KRAB 盒的单个慢病毒，通过敲除完整胰岛中感兴趣的基因，来监测仅从 β 细胞分泌胰岛素的方法。这种方法允许快速且廉价地监测来自敲除的那些 β 细胞的胰岛素分泌，从而避免了不完全胰岛感染的问题。