Liu Siming, Harata Mikako, Promes Joseph A, Burand Anthony J, Ankrum James A, Imai Yumi
Department of Internal Medicine, Carver College of Medicine, University of Iowa; Fraternal Order of Eagles Diabetes Research Center, University of Iowa.
Fraternal Order of Eagles Diabetes Research Center, University of Iowa; Roy J. Carver Department of Biomedical Engineering, University of Iowa.
J Vis Exp. 2019 May 14(147). doi: 10.3791/59578.
Various genetic tools are available to modulate genes in pancreatic islets of rodents to dissect function of islet genes for diabetes research. However, the data obtained from rodent islets are often not fully reproduced in or applicable to human islets due to well-known differences in islet structure and function between the species. Currently, techniques that are available to manipulate gene expression of human islets are very limited. Introduction of transgene into intact islets by adenovirus, plasmid, and oligonucleotides often suffers from low efficiency and high toxicity. Low efficiency is especially problematic in gene downregulation studies in intact islets, which require high efficiency. It has been known that enzymatically-dispersed islet cells reaggregate in culture forming spheroids termed pseudoislets. Size-controlled reaggregation of human islet cells creates pseudoislets that maintain dynamic first phase insulin secretion after prolonged culture and provide a window to efficiently introduce lentiviral short hairpin RNA (shRNA) with low toxicity. Here, a detailed protocol for the creation of human pseudoislets after lentiviral transduction using two commercially available multiwell plates is described. The protocol can be easily performed and allows for efficient downregulation of genes and assessment of dynamism of insulin secretion using human islet cells. Thus, human pseudoislets with lentiviral mediated gene modulation provide a powerful and versatile model to assess gene function within human islet cells.
有多种基因工具可用于调节啮齿动物胰岛中的基因，以剖析胰岛基因在糖尿病研究中的功能。然而，由于众所周知的物种间胰岛结构和功能差异，从啮齿动物胰岛获得的数据往往无法在人类胰岛中完全重现或应用于人类胰岛。目前，可用于操纵人类胰岛基因表达的技术非常有限。通过腺病毒、质粒和寡核苷酸将转基因导入完整胰岛，往往效率低下且毒性高。在完整胰岛的基因下调研究中，低效率尤其成问题，因为这类研究需要高效率。已知经酶分散的胰岛细胞在培养中重新聚集形成称为假胰岛的球体。人类胰岛细胞的大小可控重新聚集产生的假胰岛，在长时间培养后仍能维持动态的第一相胰岛素分泌，并为以低毒性高效引入慢病毒短发夹RNA（shRNA）提供了一个窗口。在此，描述了一种使用两种市售多孔板在慢病毒转导后创建人类假胰岛的详细方案。该方案易于执行，可实现基因的高效下调，并利用人类胰岛细胞评估胰岛素分泌的动态变化。因此，具有慢病毒介导基因调节的人类假胰岛为评估人类胰岛细胞内的基因功能提供了一个强大且通用的模型。
