当前位置: X-MOL 学术Indian J. Biochem. Biophys. › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
Transcriptional activation of LGR5 gene by an engineered CRISPR-Cas9-based system induces hepatic-specific factors
Indian Journal of Biochemistry and Biophysics ( IF 1.476 ) Pub Date : 2018-11-12
Dong-Ho Kim, Kye-Seong Kim, Suresh Ramakrishna

Several new approaches for reprogramming or direct reprogramming somatic cells have been implemented during the last few years. Endogenous gene activation or repression can be achieved using dead clustered regularly interspaced short palindromic repeats associated protein 9 (dCas9) system fused with a transcriptional activating or repression domain. This study was undertaken to screen and validate efficient sgRNAs targeting reprogramming or direct reprogramming transcription factors by CRISPR-Cas9 based system. In this study, we designed and validated several individual single-guide RNA (sgRNA) targeting LGR5 and Yamanaka factors, such as Oct3/4, Sox2, c-Myc and Klf4, for effective transcriptional activation in mouse cells. Furthermore, we investigated the combination of effective sgRNAs for the synchronized effect of transcriptional activation on LGR5 gene and Yamanaka factors and achieved approximately 8.4-fold and 38-fold higher levels of mouse LGR5 and Oct3/4 upregulation, respectively, compared with the control. Further, we demonstrate that the activation LGR5 gene promoter induces known defined factors responsible for direct conversion of somatic cells to hepatocyte-like cells. We envision that our validation of effective sgRNAs will facilitate the development of mouse induced pluripotent stem cells (iPSCs) and novel findings of LGR5 as a potential candidate for direct conversion of somatic cells to hepatocyte-like cells.

中文翻译:

基于CRISPR-Cas9的系统对LGR5基因的转录激活可诱导肝特异性因子

在最近几年中,已经实现了几种用于重编程或直接重编程体细胞的新方法。内源性基因的激活或抑制可以使用与转录激活或抑制域融合的死簇规则间隔的短回文重复相关蛋白9(dCas9)系统来实现。通过基于CRISPR-Cas9的系统进行了这项研究,以筛选和验证靶向重编程或直接重编程转录因子的有效sgRNA。在这项研究中,我们设计并验证了针对LGR5和Yamanaka因子(例如Oct3 / 4,Sox2,c-Myc和Klf4)的几种单个单向导RNA(sgRNA),以在小鼠细胞中进行有效的转录激活。此外,我们研究了有效的sgRNA的组合对LGR5基因和Yamanaka因子的转录激活的同步影响,与对照组相比,小鼠LGR5和Oct3 / 4上调的水平分别提高了约8.4倍和38倍。此外,我们证明激活LGR5基因启动子诱导已知的定义的因素负责体细胞直接转化为肝细胞样细胞。我们设想,我们对有效sgRNA的验证将促进小鼠诱导的多能干细胞(iPSC)的发展,以及LGR5的新发现,它是将体细胞直接转化为类肝细胞的潜在候选者。与对照相比。此外,我们证明激活LGR5基因启动子诱导已知的定义的因素负责体细胞直接转化为肝细胞样细胞。我们设想,我们对有效sgRNA的验证将促进小鼠诱导的多能干细胞(iPSC)的发展,以及LGR5的新发现,它是将体细胞直接转化为类肝细胞的潜在候选者。与对照相比。此外,我们证明激活LGR5基因启动子诱导已知的定义的因素负责体细胞直接转化为肝细胞样细胞。我们设想,我们对有效sgRNA的验证将促进小鼠诱导的多能干细胞(iPSC)的发展,以及LGR5的新发现,它是将体细胞直接转化为类肝细胞的潜在候选者。
更新日期:2018-11-12
down
wechat
bug