Generation of ΔF508-CFTR T84 cell lines by CRISPR/Cas9-mediated genome editing
Generation of ΔF508-CFTR T84 cell lines by CRISPR/Cas9-mediated genome editing
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DOI：
10.1007/s10529-016-2190-4
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发表时间：
2016-12-01
影响因子：
2.7
通讯作者：
Kim, Joo Young
中科院分区：
文献类型：
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作者：
Chung, Woo Young;Song, Myungjae;Kim, Joo Young
目的：提供一种简单的方法来制备稳定表达 Delta F508-CFTR 的 T84 细胞系，该细胞系可用作 Delta F508-CFTR 救援的有效筛选模型系统。结果：CFTR 敲除细胞系是由 Cas9 生成的，该细胞系具有靶向 CFTR 基因组外显子 1 的单引导 RNA (sgRNA)，产生的插入缺失可消除 CFTR 蛋白表达。接下来，通过慢病毒感染系统整合 Delta F508-CFTR 的基因组，实现了 Delta F508-CFTR 的稳定表达。最后，我们不仅通过在低温下培养细胞，而且在表达 Delta F508-CFTR 的已建立的 T84 细胞中与 Delta F508-CFTR 校正剂 VX-809 一起孵育，展示了 Delta F508-CFTR 的功能性拯救。结论：该细胞系统为拯救 Delta F508-CFTR 提供了一个合适的筛选平台，特别是与蛋白质折叠、逃避内质网相关蛋白质降解和膜转运相关的筛选平台。
Objectives: To provide a simple method to make a stable Delta F508-CFTR-expressing T84 cell line that can be used as an efficient screening model system for Delta F508-CFTR rescue. Results: CFTR knockout cell lines were generated by Cas9 with a single-guide RNA (sgRNA) targeting exon 1 of the CFTR genome, which produced indels that abolished CFTR protein expressions. Next, stable Delta F508-CFTR expression was achieved by genome integration of Delta F508-CFTR via the lentivirus infection system. Finally, we showed functional rescue of Delta F508-CFTR not only by growing the cells at a low temperature, but also incubating with VX-809, a Delta F508-CFTR corrector, in the established T84 cells expressing Delta F508-CFTR. Conclusions: This cell system provides an appropriate screening platform for rescue of Delta F508-CFTR, especially related to protein folding, escaped from endoplasmic-reticulum-associated protein degradation, and membrane transport.