肠内分泌细胞(EECs)仅占胃肠道表达Villin-1(Vil1)的肠上皮细胞(IECs)的一小部分;然而,总之,它们是人体最大的内分泌器官,它们中的每一个都储存和释放一组不同的肽来控制摄食行为,葡萄糖代谢,和胃肠蠕动。像所有IEC类型一样,EEC从隐窝碱基中的肠干细胞不断更新,并最终分化为成熟亚型,同时向上移动隐窝-绒毛轴。有趣的是,EEC根据其迁移状态调整其激素分泌,因为EEC沿着隐窝-绒毛轴接收改变的分化信号,从而进行功能重新适应。通过特异性启动子对成熟EEC亚型的细胞特异性靶向具有挑战性,因为EEC衍生肽及其前体的表达不仅限于EEC,而且还在其他器官中发现。例如大脑(例如,Cck和Sst)以及胰腺(例如,Sst和Gcg)。这里,我们描述了一种交叉遗传方法,该方法通过将新产生的表达Dre重组酶的小鼠品系(Vil1-2A-DD-Dre)与多种现有的Cre重组酶小鼠和小鼠品系结合起来,使细胞类型特异性靶向功能不同的EEC亚型,并带有rox和loxP位点,以进行转基因表达。我们发现,三转基因小鼠中的转基因表达在小肠末端绒毛中的I细胞而不是D和L细胞中具有高度特异性。EEC仅在末端绒毛中的靶向是由于有缺陷的2A分离肽的整合,结合低EEC内在Vil1表达,限制我们的Vil1-2A-DD-Dre小鼠品系和此处描述的交叉遗传方法仅适用于成熟EEC亚群的调查。
Enteroendocrine cells (EECs) constitute only a small proportion of Villin-1 (Vil1)-expressing intestinal epithelial cells (IECs) of the gastrointestinal tract; yet, in sum, they build the largest endocrine organ of the body, with each of them storing and releasing a distinct set of peptides for the control of feeding behavior, glucose metabolism, and gastrointestinal motility. Like all IEC types, EECs are continuously renewed from intestinal stem cells in the crypt base and terminally differentiate into mature subtypes while moving up the crypt-villus axis. Interestingly, EECs adjust their hormonal secretion according to their migration state as EECs receive altering differentiation signals along the crypt-villus axis and thus undergo functional readaptation. Cell-specific targeting of mature EEC subtypes by specific promoters is challenging because the expression of EEC-derived peptides and their precursors is not limited to EECs but are also found in other organs, such as the brain (e.g., Cck and Sst) as well as in the pancreas (e.g., Sst and Gcg). Here, we describe an intersectional genetic approach that enables cell type-specific targeting of functionally distinct EEC subtypes by combining a newly generated Dre-recombinase expressing mouse line (Vil1-2A-DD-Dre) with multiple existing Cre-recombinase mice and mouse strains with rox and loxP sites flanked stop cassettes for transgene expression. We found that transgene expression in triple-transgenic mice is highly specific in I but not D and L cells in the terminal villi of the small intestine. The targeting of EECs only in terminal villi is due to the integration of a defective 2A separating peptide that, combined with low EEC intrinsic Vil1 expression, restricts our Vil1-2A-DD-Dre mouse line and the intersectional genetic approach described here only applicable for the investigation of mature EEC subpopulations.