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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.

Genetic tool for fate mapping of Oct4 (Pou5f1)-expressing cells and their progeny past the pluripotency stage