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

Abstract

BackgroundMethods 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.MethodsThe 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).ResultsIn 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.ConclusionsThe 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.

Regulatory Elements Outside Established Pou5f1 Gene Boundaries Are Required for Multilineage Differentiation of Embryonic Stem Cells

“…Derivation of Pou5f1 ∆/∆ ;Rosa26 Pou5f1/+ ESCs was previously reported [17]. To generate Pou5f1 ∆/flox ESCs, ESCs derived from Pou5f1 flox/flox , blastocysts were seeded at a density of 5 × 10 3 per cm 2 and, with the purpose to achieve partial recombination, transiently transfected with the Cre-expressing plasmid pRosa26-TRE-CAG-Frt(Ert2CreErt2-STOP)Frt-tdTomatoPGKneo [10,24]. Twenty-four hours after transfection, the media were changed to the S/L media containing 4-hydroxytamoxifen (4-OHT).…”

“…Additionally, some Pou5f1 gene elements are important in non-pluripotent cells, for example, in the context of autoimmune diseases [6]. And while it is widely accepted that Pou5f1, particularly via the expression of the Oct4 protein isoform A, plays important roles exclusively in pluripotent stem cells and cells of germline lineage [1,7], new data challenging this paradigm have been emerging, further emphasizing the necessity of studying Pou5f1 regulation [8][9][10].…”

Human AlphoidtetO Artificial Chromosome as a Gene Therapy Vector for the Developing Hemophilia A Model in Mice

“…Lentiviruses in the cell culture supernatant were prepared as previously described [30][31][32][33]. Lentiviruses encoding either the polycistronic cassette containing pluripotency factors Oct4, Sox2, Klf4, cMyc, or rtTA, or Env∆R were prepared [34,35].…”

“…Teratoma analysis was performed as described previously [25,33]. Cells were trypsinized, washed with PBS, and then 10 6 cells were injected subcutaneously in the hindlimb of NUDE mice.…”

“…The cells were grown in 48-well plates, washed with PBS, and fixed in 4% paraformaldehyde in PBS for 10 min [33,40]. The fixed cells were washed with PBS, permeabilized by 0,1% Triton X-100 solution in PBS for 15 min, and incubated with 3% bovine serum albumin (BSA) in PBS for 1 h at RT.…”