Probing expression of E-selectin using CRISPR-Cas9-mediated tagging with HiBiT in human endothelial cells.
Probing expression of E-selectin using CRISPR-Cas9-mediated tagging with HiBiT in human endothelial cells.
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DOI：
10.1016/j.isci.2023.107232
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发表时间：
2023-07-21
期刊：
影响因子：
5.8
通讯作者：
Kilpatrick, Laura E.
中科院分区：
文献类型：
--
作者：
Ogrodzinski, Lydia;Platt, Simon;Goulding, Joelle;Alexander, Cameron;Farr, Tracy D.;Woolard, Jeanette;Hill, Stephen J.;Kilpatrick, Laura E.
E-选择素在内皮细胞上表达以响应炎性细胞因子并介导白细胞滚动和外渗。然而，由于缺乏实验方法来监测活细胞中真实的表达，研究受到阻碍。在此，NanoLuc二元技术（NanoBiT）结合CRISPR-Cas9基因组编辑用于用11个氨基酸的纳米荧光素酶片段HiBiT标记人脐静脉内皮细胞（HUVEC）中的内源性E-选择素。添加膜不可渗透的互补片段LgBiT允许检测细胞表面表达。这使得炎症介质对E-选择素表达的影响能够在活的内皮细胞中真实的时间内被监测。NanoBiT结合CRISPR-Cas9基因编辑可以灵敏地监测E-选择素细胞表面表达的实时变化，并为未来针对这种重要炎症蛋白的药物发现工作提供了强大的工具。CRISPR-Cas9编辑将HiBiT添加到HUVEC中E-选择素的N末端添加纯化的LgBiT允许纳米荧光素酶的再互补然后监测E-选择素的细胞表面表达的实时变化分子生物学;生物技术;细胞生物学
E-selectin is expressed on endothelial cells in response to inflammatory cytokines and mediates leukocyte rolling and extravasation. However, studies have been hampered by lack of experimental approaches to monitor expression in real time in living cells. Here, NanoLuc Binary Technology (NanoBiT) in conjunction with CRISPR-Cas9 genome editing was used to tag endogenous E-selectin in human umbilical vein endothelial cells (HUVECs) with the 11 amino acid nanoluciferase fragment HiBiT. Addition of the membrane-impermeable complementary fragment LgBiT allowed detection of cell surface expression. This allowed the effect of inflammatory mediators on E-selectin expression to be monitored in real time in living endothelial cells. NanoBiT combined with CRISPR-Cas9 gene editing allows sensitive monitoring of real-time changes in cell surface expression of E-selectin and offers a powerful tool for future drug discovery efforts aimed at this important inflammatory protein. E-selectin is expressed on endothelial cells in response to inflammatory cytokines CRISPR-Cas9 editing has added HiBiT to the N terminus of E-selectin in HUVECs Addition of purified LgBiT allows re-complementation of the nanoluciferase enzyme Real-time changes in cell surface expression of E-selectin have then be monitored Molecular biology; Biotechnology; Cell biology
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影响因子：
4.8
作者：
Kim, I;Moon, SO;Koh, GY
通讯作者：
Koh, GY
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作者：
Kappelmayer J;Nagy B Jr
通讯作者：
Nagy B Jr
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作者：
Shamay, Yosi;Raviv, Lior;David, Ayelet
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46.9
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影响因子：
4.8
作者：
Read, MA;Whitley, MZ;Collins, T
通讯作者：
Collins, T