Abstract：
Androglobin (ADGB), the most recently identified member of the mammalian globin family, is a chimeric protein with an unusual, embedded globin domain that is circularly permutated and exhibits hallmarks of a hexacoordinated heme-binding scheme. Whereas abundant expression of ADGB was initially found to be mainly restricted to cells in the postmeiotic stages of spermatogenesis, more recent RNA-Seq-based expression analysis data revealed that ADGB is detectable in cells carrying motile cilia or flagella. This very tight regulation of ADGB gene expression urges the need for alternative techniques to study endogenous expression in classical mammalian cell models, which do not express ADGB. We describe here the use of CRISPR activation (CRISPRa) technology to induce endogenous ADGB gene expression in HEK293T, MCF-7, and HeLa cells from its promoter and illustrate how this method can be employed to validate putative regulatory DNA elements of ADGB in promoter and enhancer regions.
摘要：
雄红蛋白（ADGB），哺乳动物珠蛋白家族的最新成员，是一种嵌合蛋白,嵌入的珠蛋白域，进行循环置换，并表现出六配位血红素结合方案的标志。尽管最初发现ADGB的大量表达主要限于精子发生减数分裂后阶段的细胞，最近的基于RNA-Seq的表达分析数据显示，ADGB可在携带活动纤毛或鞭毛的细胞中检测到。这种对ADGB基因表达的非常严格的调控促使需要替代技术来研究经典哺乳动物细胞模型中的内源性表达。不表示ADGB。我们在这里描述了使用CRISPR激活（CRISPRa）技术在HEK293T中诱导内源性ADGB基因表达，MCF-7和HeLa细胞从其启动子，并说明如何使用该方法来验证启动子和增强子区域中ADGB的推定调节DNA元件。
