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Genetic tool for fate mapping of Oct4 (Pou5f1)-expressing cells and their progeny past the pluripotency stage.
Stem Cell Research & Therapy ( IF 7.5 ) Pub Date : 2019-12-16 , DOI: 10.1186/s13287-019-1520-6
Andrey A Kuzmin 1 , Veronika V Ermakova 1 , Sergey A Sinenko 1 , Sergey V Ponomartsev 1 , Tatiana Y Starkova 1 , Elena V Skvortsova 1 , Olga Cherepanova 2 , Alexey N Tomilin 1, 3
Affiliation  

BACKGROUND Methods based on site-specific recombinases are widely used in studying gene activities in vivo and in vitro. In these studies, constitutively active or inducible variants of these recombinases are expressed under the control of either lineage-specific or ubiquitous promoters. However, there is a need for more advanced schemes that combine these features with possibilities to choose a time point from which lineage tracing starts in an autonomous fashion. For example, the key mammalian germline gatekeeper gene Oct4 (Pou5f1) is expressed in the peri-implantation epiblast which gives rise to all cells within embryos. Thus the above techniques are hardly applicable to Oct4 tracing past the epiblast stage, and the establishment of genetic tools addressing such a limitation is a highly relevant pursuit. METHODS The CRISPR/Cas9 tool was used to manipulate the genome of mouse embryonic stem cells (ESCs), and various cell culture technics-to maintain and differentiate ESCs to neural cell, lentivirus-based reprogramming technique-to generate induced pluripotent stem cells (iPSCs). RESULTS In this paper, we have developed a two-component genetic system (referred to as O4S) that allows tracing Oct4 gene activity past the epiblast stage of development. The first component represents a knock-in of an ubiquitous promoter-driven inducible Cre, serving as a stop signal for downstream tdTomato. Upon activation of Cre activity with 4-hydroxytamoxifen (4-OHT) at any given time point, the recombinase excises a stop signal and poses the second component of the system-the FlpO recombinase, knocked into 3'UTR of Oct4, to be expressed upon activation of the latter gene. Oct4-driven expression of FlpO, in turn, triggers the tdTomato expression and thus, permanently marks Oct4+ cells and their progeny. We have validated the O4S system in cultured ESCs and shown that it is capable, for example, to timely capture an activation of Oct4 gene during the reprogramming of somatic cells into iPSCs. CONCLUSIONS The developed O4S system can be used to detect Oct4 activation event, both permanent and transient, in somatic cell types outside the germline. The approach can be equally adjusted to other genes, provided the first component of the system is placed under transcriptional control of these genes, thus, making it a valuable tool for cell fate mapping in mice.

中文翻译:

遗传工具,用于确定表达Oct4(Pou5f1)的细胞及其后代经过多能性阶段的命运。

背景技术基于位点特异性重组酶的方法被广泛用于研究体内和体外的基因活性。在这些研究中,这些重组酶的组成型活性或诱导型变体在谱系特异性或普遍存在的启动子的控制下表达。但是,需要更高级的方案,这些方案将这些功能与选择时间点的可能性结合起来,从该时间点以自动方式开始沿袭。例如,关键的哺乳动物种系看门人基因Oct4(Pou5f1)在植入前的成骨细胞中表达,从而产生了胚胎内的所有细胞。因此,以上技术几乎不适用于成骨细胞阶段后的Oct4追踪,解决这种局限性的遗传工具的建立是高度相关的追求。方法使用CRISPR / Cas9工具操纵小鼠胚胎干细胞(ESC)的基因组,并采用多种细胞培养技术-维持和区分ESC至基于慢病毒的重编程神经细胞技术-产生诱导性多能干细胞(iPSC) )。结果在本文中,我们开发了一个两成分的遗传系统(称为O4S),该系统可以追踪发育上皮阶段的Oct4基因活性。第一个成分代表了普遍存在的启动子驱动的诱导型Cre的敲入,充当下游tdTomato的终止信号。在任何给定时间点用4-羟基他莫昔芬(4-OHT)激活Cre活性后,重组酶均会切除一个终止信号,并构成系统的第二个成分-FlpO重组酶,敲入Oct4的3'UTR,在激活后一个基因时表达。Oct4驱动的FlpO表达继而触发tdTomato表达,从而永久标记Oct4 +细胞及其后代。我们已经在培养的ESC中验证了O4S系统,并表明它能够例如在将体细胞重编程为iPSC的过程中及时捕获Oct4基因的激活。结论所开发的O4S系统可用于检测种系外体细胞类型中的Oct4激活事件,包括永久性的和短暂的。如果将系统的第一个组件置于这些基因的转录控制之下,则该方法可以同等地调整为其他基因,因此,使其成为在小鼠中进行细胞命运定位的有价值的工具。永久标记Oct4 +细胞及其后代。我们已经在培养的ESC中验证了O4S系统,并表明它能够例如在将体细胞重编程为iPSC的过程中及时捕获Oct4基因的激活。结论所开发的O4S系统可用于检测种系外体细胞类型中的Oct4激活事件,包括永久性的和短暂的。如果将系统的第一个组件置于这些基因的转录控制之下,则该方法可以同等地调整为其他基因,因此,使其成为在小鼠中进行细胞命运定位的有价值的工具。永久标记Oct4 +细胞及其后代。我们已经在培养的ESC中验证了O4S系统,并表明它能够例如在将体细胞重编程为iPSC的过程中及时捕获Oct4基因的激活。结论所开发的O4S系统可用于检测种系外体细胞类型中的Oct4激活事件,包括永久性的和短暂的。如果将系统的第一个组件置于这些基因的转录控制之下,则该方法可以同等地调整为其他基因,因此,使其成为在小鼠中进行细胞命运定位的有价值的工具。结论所开发的O4S系统可用于检测种系外体细胞类型中的Oct4激活事件,包括永久性的和短暂的。如果将系统的第一个组件置于这些基因的转录控制之下,则该方法可以同等地调整为其他基因,因此,使其成为在小鼠中进行细胞命运定位的有价值的工具。结论所开发的O4S系统可用于检测种系外体细胞类型中的Oct4激活事件,包括永久性的和短暂的。如果将系统的第一个组件置于这些基因的转录控制之下,则该方法可以同等地调整为其他基因,因此,使其成为在小鼠中进行细胞命运定位的有价值的工具。
更新日期:2019-12-16
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