Inducible Cassette Exchange: A Rapid and Efficient System Enabling Conditional Gene Expression in Embryonic Stem and Primary Cells

Iacovino, Michelina; Bosnakovski, Darko; Fey, Holger; Rux, Danielle; Bajwa, Gagan; Mahen, Elisabeth; Mitanoska, Ana; Xu, Zhaohui; Kyba, Michael

doi:10.1002/stem.715

Cited by 175 publications

(220 citation statements)

References 30 publications

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“…In these conditions, most of the cells differentiated spontaneously into a neural fate [29][30][31][32]. To study the role of Eomes, we generated a recombinant ESC line, in which the expression of a Myc-tagged version of mouse Eomes can be induced on doxycyclin (Dox) addition [33,34] (Fig 1A and supplementary Fig S1 online).…”

Section: Results and Discussion Eomes Promotes Cardiogenesis In Escmentioning

confidence: 99%

“…After electroporation into A2Lox.Cre cells and cassette exchange recombination [33], neomycin-resistant clones were screened for their expression of Myc-Eomes or Flag-Mesp1-Engrailed by immunofluorescence (supplementary Fig S1 online) and qRT-PCR (data not shown) 24 h after induction. Results obtained with the Myc-Eomes ESC line were confirmed in three independent clones.…”

Section: Methodsmentioning

confidence: 99%

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Eomesodermin induces Mesp1 expression and cardiac differentiation from embryonic stem cells in the absence of Activin

et al. 2012

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The transcription factor Eomesodermin (Eomes) is involved in early embryonic patterning, but the range of cell fates that it controls as well as its mechanisms of action remain unclear. Here we show that transient expression of Eomes promotes cardiovascular fate during embryonic stem cell differentiation. Eomes also rapidly induces the expression of Mesp1, a key regulator of cardiovascular differentiation, and directly binds to regulatory sequences of Mesp1. Eomes effects are strikingly modulated by Activin signalling: high levels of Activin inhibit the promotion of cardiac mesoderm by Eomes, while they enhance Eomes-dependent endodermal specification. These results place Eomes upstream of the Mesp1-dependent programme of cardiogenesis, and at the intersection of mesodermal and endodermal specification, depending on the levels of Activin/Nodal signalling.

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Section: Results and Discussion Eomes Promotes Cardiogenesis In Escmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Eomesodermin induces Mesp1 expression and cardiac differentiation from embryonic stem cells in the absence of Activin

et al. 2012

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“…4A and Fig. S5) (15). The expression of Isl1-Lhx3 was robustly induced by doxycycline (Dox) treatment (Fig.…”

Section: Isl1-lhx3 Efficiently Directs Differentiation Of Mouse Escs mentioning

confidence: 91%

Fusion protein Isl1–Lhx3 specifies motor neuron fate by inducing motor neuron genes and concomitantly suppressing the interneuron programs

Lee

Cuvillier

Lee

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Combinatorial transcription codes generate the myriad of cell types during development and thus likely provide crucial insights into directed differentiation of stem cells to a specific cell type. The LIM complex composed of Isl1 and Lhx3 directs the specification of spinal motor neurons (MNs) in embryos. Here, we report that Isl1-Lhx3, a LIM-complex mimicking fusion, induces a signature of MN transcriptome and concomitantly suppresses interneuron differentiation programs, thereby serving as a potent and specific inducer of MNs in stem cells. We show that an equimolar ratio of Isl1 and Lhx3 and the LIM domain of Lhx3 are crucial for generating MNs without up-regulating interneuron genes. These led us to design Isl1-Lhx3, which maintains the desirable 1:1 ratio of Isl1 and Lhx3 and the LIM domain of Lhx3. Isl1-Lhx3 drives MN differentiation with high specificity and efficiency in the spinal cord and embryonic stem cells, bypassing the need for sonic hedgehog (Shh). RNA-seq analysis revealed that Isl1-Lhx3 induces the expression of a battery of MN genes that control various functional aspects of MNs, while suppressing key interneuron genes. Our studies uncover a highly efficient method for directed MN generation and MN gene networks. Our results also demonstrate a general strategy of using embryonic transcription complexes for producing specific cell types from stem cells. D eveloping central nervous system (CNS) produces a vast number of neuronal types, but adult CNS has only limited capacity to regenerate neurons. This has prompted great interest in identifying methods to produce specific neuronal types from stem cells. Production of differentiated cell types from pluripotent stem cells, such as embryonic stem cells (ESCs), should enable a continuous supply of diseased cell types for drug screening and cell replacement therapy and provide valuable insights into the pathophysiology of human diseases. One important challenge in this effort is to steer stem cells into specific cell types. Recapitulation of normal developmental processes using embryonic inductive signals has been used to drive differentiation of pluripotent stem cells into specific cell types (1). However, this strategy tends to trigger formation of mixed cell types rather than a targeted cell type, because each inductive signal is used in multiple developmental pathways. This shortcoming might be circumvented by using more specific, downstream transcription factors of inductive signals. In this regard, it should be noted that many transcription factors function in combination to determine cell fates during development, suggesting that coexpression of multiple transcription factors could be a more effective method to generate a particular cell type from pluripotent stem cells.Motor neurons (MNs) in the spinal cord project axons to muscles and control their contraction. The developmental pathways to generate MNs have been relatively well studied. In the developing spinal cord, sonic hedgehog (Shh) signal triggers the expression of two LIM homeodomain...

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“…Dox-inducible ESC lines were generated using the A2.Lox.Cre inducible cassette exchange system (36). Briefly, 1x and 3xFlag epitope, N-terminally tagged Sp5, Sp8, and Lef1 cDNA's were PCR cloned into the MluI/AflII restriction sites of the P2.Lox vector.…”

Section: Methodsmentioning

confidence: 99%

Sp5 and Sp8 recruit β-catenin and Tcf1-Lef1 to select enhancers to activate Wnt target gene transcription

Kennedy

Chalamalasetty

Thomas

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The ancient, highly conserved, Wnt signaling pathway regulates cell fate in all metazoans. We have previously shown that combined null mutations of the specificity protein (Sp) 1/Klf-like zinc-finger transcription factors Sp5 and Sp8 (i.e., Sp5/8) result in an embryonic phenotype identical to that observed when core components of the Wnt/β-catenin pathway are mutated; however, their role in Wnt signal transduction is unknown. Here, we show in mouse embryos and differentiating embryonic stem cells that Sp5/8 are gene-specific transcriptional coactivators in the Wnt/β-catenin pathway. Sp5/8 bind directly to GC boxes in Wnt target gene enhancers and to adjacent, or distally positioned, chromatin-bound T-cell factor (Tcf) 1/lymphoid enhancer factor (Lef) 1 to facilitate recruitment of β-catenin to target gene enhancers. Because Sp5 is itself directly activated by Wnt signals, we propose that Sp5 is a Wnt/β-catenin pathway-specific transcripton factor that functions in a feed-forward loop to robustly activate select Wnt target genes.S ignaling pathways in multicellular organisms have evolved over millions of years to accommodate complex programs of tissue-specific gene expression. One such pathway, the Wnt/ β-catenin pathway, regulates gene expression by elevating the cytosolic levels of the transcription coactivator β-catenin (1). Stabilized β-catenin translocates to the nucleus, where it interacts with the DNA-bending, DNA-binding Tcf1 and Lef1 transcription factors (TFs), which subsequently replace Groucho/ Tcf3 repressor complexes on Wnt target gene enhancers (2). β-Catenin interacts with cell context-dependent cofactors (web. stanford.edu/group/nusselab/cgi-bin/wnt/) to associate with RNA polymerase II and the general transcription apparatus to activate transcription. However, the nature of the β-cateninTcf/Lef enhancer-binding protein complex and the mechanisms that facilitate its association with regulatory elements at Wnt target genes remain poorly understood.The formation of a Wnt signaling center during gastrulation is essential for animal development (3). Secreted Wnts emanating from the primitive streak regulate the fate of posterior progenitors, including the neuromesodermal progenitor (NMP), an embryonic cell that depends upon Wnt3a for self-renewal and mesodermal differentiation and that gives rise to the spinal cord, dermis, and musculoskeletal system of the trunk and tail (4-6). Embryos lacking Wnt3a, Ctnnb1 (β-catenin), T-cell factor (Tcf) 1 and lymphoid enhancer factor (Lef) 1, or specificity protein (Sp) 5 and Sp8 display similar severe posterior truncations caused by the loss of NMPs (7-10). These genes define a syn-phenotype group that, together with the genetic interactions observed between Sp5 and Wnt3a, suggests that Sp5/8 could be effectors of Wnt signaling. Sp5/8 are closely related to Sp1, which is one of the first identified eukaryotic TFs (11) and is frequently associated with the regulation of housekeeping genes. In contrast to the ubiquitously expressed Sp1, Sp5 expression is restricte...

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Inducible Cassette Exchange: A Rapid and Efficient System Enabling Conditional Gene Expression in Embryonic Stem and Primary Cells

Cited by 175 publications

References 30 publications

Eomesodermin induces Mesp1 expression and cardiac differentiation from embryonic stem cells in the absence of Activin

Eomesodermin induces Mesp1 expression and cardiac differentiation from embryonic stem cells in the absence of Activin

Fusion protein Isl1–Lhx3 specifies motor neuron fate by inducing motor neuron genes and concomitantly suppressing the interneuron programs

Sp5 and Sp8 recruit β-catenin and Tcf1-Lef1 to select enhancers to activate Wnt target gene transcription

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