Pauerstein Philip T, Park Keon Min, Peiris Heshan S, Wang Jing, Kim Seung K
Department of Developmental Biology (P.T.P., K.M.P., H.S.P., J.W., S.K.K.) and Howard Hughes Medical Institute (S.K.K.), Stanford University School of Medicine, Stanford, California 94305.
Mol Endocrinol. 2016 Feb;30(2):248-53. doi: 10.1210/me.2015-1220. Epub 2016 Jan 8.
The 2 most abundant human pancreatic islet cell types are insulin-producing β-cells and glucagon-producing α-cells. Defined cis-regulatory elements from rodent Insulin genes have permitted genetic labeling of human islet β-cells, enabling lineage tracing and generation of human β-cell lines, but analogous elements for genetically labeling human α-cells with high specificity do not yet exist. To identify genetic elements that specifically direct reporter expression to human α-cells, we investigated noncoding sequences adjacent to the human GLUCAGON and ARX genes, which are expressed in islet α-cells. Elements with high evolutionary conservation were cloned into lentiviral vectors to direct fluorescent reporter expression in primary human islets. Based on the specificity of reporter expression for α- and β-cells, we found that rat glucagon promoter was not specific for human α-cells but that addition of human GLUCAGON untranslated region sequences substantially enhanced specificity of labeling in both cultured and transplanted islets to a degree not previously reported, to our knowledge. Specific transgene expression from these cis-regulatory sequences in human α-cells should enable targeted genetic modification and lineage tracing.
人类胰腺中最丰富的两种胰岛细胞类型是产生胰岛素的β细胞和产生胰高血糖素的α细胞。来自啮齿动物胰岛素基因的特定顺式调控元件已可对人类胰岛β细胞进行基因标记,从而实现谱系追踪和人类β细胞系的生成,但尚未存在用于以高特异性对人类α细胞进行基因标记的类似元件。为了鉴定能将报告基因表达特异性导向人类α细胞的遗传元件,我们研究了与在胰岛α细胞中表达的人类胰高血糖素(GLUCAGON)基因和无尾同源盒基因(ARX)相邻的非编码序列。具有高度进化保守性的元件被克隆到慢病毒载体中,以在原代人类胰岛中指导荧光报告基因的表达。基于报告基因对α细胞和β细胞表达的特异性,我们发现大鼠胰高血糖素启动子对人类α细胞不具有特异性,但添加人类胰高血糖素非翻译区序列可在培养的和移植的胰岛中大幅提高标记特异性,据我们所知,其程度是此前未曾报道过的。这些顺式调控序列在人类α细胞中的特异性转基因表达应能实现靶向基因修饰和谱系追踪。