Mutations in more than half of human connexin genes encoding gap junction (GJ) subunits have been linked to inherited human diseases. Functional studies of human GJ channels are essential for revealing mechanistic insights into the etiology of disease-linked connexin mutants. However, the commonly used Xenopus oocytes, N2A, HeLa, and other model cells for recombinant expression of human connexins have different and significant limitations. Here we developed a human cell line (HEK293) with each of the endogenous connexins (Cx43 and Cx45) knocked out using the CRISPR-Cas9 system. Double knockout HEK293 cells showed no background GJ coupling, were easily transfected with several human connexin genes (such as those encoding Cx46, Cx50, Cx37, Cx45, Cx26, and Cx36) which successfully formed functional GJs and were readily accessible for dual patch clamp analysis. Single knockout Cx43 or Cx45 HEK cell lines could also be used to characterize human GJ channels formed by Cx45 or Cx43, respectively, with an expression level suitable for studying macroscopic and single channel GJ channel properties. A cardiac arrhythmia linked Cx45 mutant R184G failed to form functional GJs in DKO HEK293 cells with impaired localizations. These genetically engineered HEK293 cells are well suited for patch clamp study of human GJ channels.

中文翻译：

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基因工程人胚胎肾细胞作为人类间隙连接通道双膜片钳研究的新型载体


超过一半的编码间隙连接（GJ）亚基的人类连接蛋白基因的突变与人类遗传性疾病有关。人类 GJ 通道的功能研究对于揭示与疾病相关的连接蛋白突变体的病因学机制至关重要。然而，常用的非洲爪蟾卵母细胞、N2A、HeLa 和其他用于重组表达人类连接蛋白的模型细胞具有不同且显着的局限性。在这里，我们开发了一种人类细胞系 (HEK293)，其中每个内源连接蛋白（Cx43 和 Cx45）均使用 CRISPR-Cas9 系统敲除。双敲除 HEK293 细胞没有表现出背景 GJ 偶联，可以轻松转染多个人连接蛋白基因（例如编码 Cx46、Cx50、Cx37、Cx45、Cx26 和 Cx36 的基因），成功形成功能性 GJ，并且易于进行双膜片钳分析。单敲除 Cx43 或 Cx45 HEK 细胞系也可用于表征分别由 Cx45 或 Cx43 形成的人 GJ 通道，其表达水平适合研究宏观和单通道 GJ 通道特性。与心律失常相关的 Cx45 突变体 R184G 无法在定位受损的 DKO HEK293 细胞中形成功能性 GJ。这些基因工程 HEK293 细胞非常适合人类 GJ 通道的膜片钳研究。