State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Animal Reproduction Institute, Guangxi University, Nanning, Guangxi, China.
Department of Animal Science, Institute for Systems Genomics, University of Connecticut, Storrs, CT, United States of America.
PLoS One. 2018 Oct 5;13(10):e0203923. doi: 10.1371/journal.pone.0203923. eCollection 2018.
Green fluorescent protein (GFP) reporters controlled by the regulatory region of OCT4 and NANOG-two master regulators for pluripotency are widely used in studies of pluripotent stem cell establishment and embryo development. Alongside the challenge in establishing bovine pluripotent stem cells, the application of bovine-specific gene reporters has rarely been explored. Using lentivirus-based GFP reporter, we investigated the upstream regulatory regions of bovine OCT4 and NANOG. These reporters show activity in both naïve- and primed-state pluripotency when infected into mouse and human embryonic stem cells (ESCs), respectively. Consistent with what is found in humans and mice, the bovine OCT4-distal enhancer (bOCT4-DE) but not the proximal enhancer (bOCT4-PE) region is preferentially activated in naïve-state pluripotency. Furthermore, the bOCT4-DE region is silenced upon conversion of naive-state ESCs into primed-state epiblast stem cells (EpiSCs). Co-infection of mouse fibroblasts with the reprograming factors for induced pluripotent stem cell (iPSC) induction leads to the generation of GFP positive colonies, demonstrating that these GFP reporters can serve as live indicators for induced pluripotent cell establishment. We further proved that the bovine OCT4 distal enhancer is active in bovine blastocysts. We established the lentiviral-based fluorescent reporters controlled by bovine OCT4 and NANOG enhancer sequences. These reporter constructs show activity in naïve- and primed-pluripotent states. These reporters may serve as versatile tools for bovine ESC/iPSC generation and identification, as well as for developmental studies of bovine embryos.
绿色荧光蛋白（GFP）报告基因受多能性调控因子 OCT4 和 NANOG 的调控区控制，广泛用于多能干细胞建立和胚胎发育的研究。在建立牛多能干细胞的挑战的同时，牛特异性基因报告基因的应用很少被探索。我们使用基于慢病毒的 GFP 报告基因，研究了牛 OCT4 和 NANOG 的上游调控区。这些报告基因在感染小鼠和人胚胎干细胞（ESCs）时分别在原始和诱导多能性中具有活性。与在人和小鼠中发现的情况一致，牛 OCT4 远端增强子（bOCT4-DE）而不是近端增强子（bOCT4-PE）区域在原始多能性中优先被激活。此外，bOCT4-DE 区域在原始态 ESCs 转化为诱导多能态外胚层干细胞（EpiSCs）时被沉默。将重编程因子共同感染小鼠成纤维细胞以诱导多能干细胞（iPSC）诱导，会导致 GFP 阳性集落的产生，表明这些 GFP 报告基因可作为诱导多能细胞建立的活体指示剂。我们进一步证明牛 OCT4 远端增强子在牛胚泡中具有活性。我们建立了受牛 OCT4 和 NANOG 增强子序列控制的基于慢病毒的荧光报告基因。这些报告基因构建体在原始和诱导多能态中具有活性。这些报告基因可能成为牛 ESC/iPSC 生成和鉴定以及牛胚胎发育研究的多功能工具。
