Generation of a knock-in MAP2-tdTomato reporter human embryonic stem cell line with inducible expression of NEUROG2/1 (NYGCe001-A).
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ABSTRACT:
SUBMITTER: Lu C
PROVIDER: S-EPMC6919562 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The porcine pluripotent cells that can generate germline chimeras have not been developed. The Oct4 promoter-based fluorescent reporter system, which can be used to monitor pluripotency, is an important tool to generate authentic porcine pluripotent cells. In this study, we established a porcine Oct4 reporter system, wherein the endogenous Oct4 promoter directly controls red fluorescent protein (RFP). 2A-tdTomato sequence was inserted to replace the stop codon of the porcine Oct4 gene by homogenous recombination (HR). Thus, the fluorescence can accurately show the activation of endogenous Oct4. Porcine fetal fibroblast (PFF) lines with knock-in (KI) of the tdTomato gene in the downstream of endogenous Oct4 promoter were achieved using the CRISPR/CAS9 system. Transgenic PFFs were used as donor cells for somatic cell nuclear transfer (SCNT). Strong RFP expression was detected in the blastocysts and genital ridges of SCNT fetuses but not in other tissues. Two viable transgenic piglets were also produced by SCNT. Reprogramming of fibroblasts from the fetuses and piglets by another round of SCNT resulted in tdTomato reactivation in reconstructed blastocysts. Result indicated that a KI porcine reporter system to monitor the pluripotent status of cells was successfully developed.
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Project description:Copper transporter 1 (CTR1) is the major membrane protein responsible for cellular copper (Cu) uptake and mediates cellular copper homeostasis. To elucidate CTR1's behavior using imaging approaches, we generated a homozygous knock-in human embryonic stem cell (hESC) clone expressing photoconvertible fluorescence protein mEos4b-tagged endogenous CTR1 using CRISPR-Cas9 mediated homologous recombination. The engineered cells express functional CTR1-mEos4b fusion and have normal stem cell morphology. They remain pluripotent and can be differentiated into all three germ layers in vitro. This resource allows the study of CTR1 at an endogenous level in different cellular contexts using microscopy.
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Project description:Mouse parthenogenetic haploid embryonic stem cells (ESCs) are pluripotent cells generated from chemically activated oocytes. Haploid ESCs provide an opportunity to study the effect of genetic alterations because of their hemizygotic characteristics. However, their further application for the selection of unique phenotypes remains limited since ideal reporters to monitor biological processes such as cell differentiation are missing. Here, we report the application of CRISPR/Cas9-mediated knock-in of a reporter cassette, which does not disrupt endogenous target genes in mouse haploid ESCs. We first validated the system by inserting the P2A-Venus reporter cassette into the housekeeping gene locus. In addition to the conventional strategy using the Cas9 nuclease, we employed the Cas9 nickase and truncated sgRNAs to reduce off-target mutagenesis. These strategies induce targeted insertions with an efficiency that correlated with sgRNA guiding activity. We also engineered the neural marker gene Sox1 locus and verified the precise insertion of the P2A-Venus reporter cassette and its functionality by monitoring neural differentiation. Our data demonstrate the successful application of the CRISPR/Cas9-mediated knock-in system for establishing haploid knock-in ESC lines carrying gene specific reporters. Genetically modified haploid ESCs have potential for applications in forward genetic screening of developmental pathways.
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Project description:BackgroundThe pluripotent state in embryonic stem (ES) cells is controlled by a core network of transcription factors that includes Nanog, Oct4 and Sox2. Nanog is required to reach pluripotency during somatic reprogramming and is the only core factor whose overexpression is able to oppose differentiation-promoting signals. Additionally, Nanog expression is known to fluctuate in ES cells, and different levels of Nanog seem to correlate with ES cells' ability to respond to differentiation promoting signals. Elucidating how dynamic Nanog levels are regulated in pluripotent cells and modulate their potential is therefore critical to develop a better understanding of the pluripotent state.Methodology/principal findingsWe describe the generation and validation of a mouse ES cell line with a novel Nanog reporter (Nd, from Nanog dynamics), containing a BAC transgene where the short-lived fluorescent protein VNP is placed under Nanog regulation. We show that Nanog and VNP have similar half-lives, and that Nd cells provide an accurate and measurable read-out for the dynamic levels of Nanog. Using this reporter, we could show that ES cells with low Nanog levels indeed have higher degree of priming to differentiation, when compared with high-Nanog cells. However, low-Nanog ES cells maintain high levels of Oct4 and Sox2 and can revert to a state of high-Nanog expression, indicating that they are still within the window of pluripotency. We further show that the observed changes in Nanog levels correlate with ES cell morphology and that Nanog dynamic expression is modulated by the cellular environment.Conclusions/significanceThe novel reporter ES cell line here described allows an accurate monitoring of Nanog's dynamic expression in the pluripotent state. This reporter will thus be a valuable tool to obtain quantitative measurements of global gene expression in pluripotent ES cells in different states, allowing a detailed molecular mapping of the pluripotency landscape.
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Project description:Gonadotropin-releasing hormone (GnRH) neurons provide a fundamental signal for the onset of puberty and subsequent reproductive functions by secretion of gonadotropin-releasing hormone. Their disrupted development or function leads to congenital hypogonadotropic hypogonadism (CHH). To model the development of human GnRH neurons, we generated a stable GNRH1-TdTomato reporter cell line in human pluripotent stem cells (hPSCs) using CRISPR-Cas9 genome editing. RNA-sequencing of the reporter clone, differentiated into GnRH neurons by dual SMAD inhibition and FGF8 treatment, revealed 6461 differentially expressed genes between progenitors and GnRH neurons. Expression of the transcription factor ISL1, one of the top 50 most upregulated genes in the TdTomato-expressing GnRH neurons, was confirmed in 10.5 gestational week-old human fetal GnRH neurons. Among the differentially expressed genes, we detected 15 genes that are implicated in CHH and several genes that are implicated in human puberty timing. Finally, FGF8 treatment in the neuronal progenitor pool led to upregulation of 37 genes expressed both in progenitors and in TdTomato-expressing GnRH neurons, which suggests upstream regulation of these genes by FGF8 signaling during GnRH neuron differentiation. These results illustrate how hPSC-derived human GnRH neuron transcriptomic analysis can be utilized to dissect signaling pathways and gene regulatory networks involved in human GnRH neuron development.This article has an associated First Person interview with the first author of the paper.
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Project description:BackgroundRefinement of therapeutic-scale platelet production in vitro will provide a new source for transfusion in patients undergoing chemotherapy or radiotherapy. However, procedures for cost-effective and scalable platelet generation remain to be established.MethodsIn this study, we established human embryonic stem cell (hESC) lines containing knock-in of thrombopoietin (TPO) via CRISPR/Cas9-mediated genome editing. The expression and secretion of TPO was detected by western blotting and enzyme-linked immunosorbent assay. Then, we tested the potency for hematopoietic differentiation by coculturing the cells with mAGM-S3 cells and measured the generation of CD43+ and CD45+ hematopoietic progenitor cells (HPCs). The potency for megakaryocytic differentiation and platelet generation of TPO knock-in hESCs were further detected by measuring the expression of CD41a and CD42b. The morphology and function of platelets were analyzed with electronic microscopy and aggregation assay.ResultsThe TPO gene was successfully inserted into the AAVS1 locus of the hESC genome and two cell lines with stable TPO expression and secretion were established. TPO knock-in exerts minimal effects on pluripotency but enhances early hematopoiesis and generation of more HPCs. More importantly, upon its knock-in, TPO augments megakaryocytic differentiation and platelet generation. In addition, the platelets derived from hESCs in vitro are functionally and morphologically comparable to those found in peripheral blood. Furthermore, TPO knock-in can partially replace the large quantities of extrinsic TPO necessary for megakaryocytic differentiation and platelet generation.ConclusionsOur results demonstrate that autonomous production of cytokines in hESCs may become a powerful approach for cost-effective and large-scale platelet generation in translational medicine.
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Project description: Not available
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Project description:AimsHuman embryonic stem cells (hESCs) can be used to generate scalable numbers of cardiomyocytes (CMs) for studying cardiac biology, disease modelling, drug screens, and potentially for regenerative therapies. A fluorescence-based reporter line will significantly enhance our capacities to visualize the derivation, survival, and function of hESC-derived CMs. Our goal was to develop a reporter cell line for real-time monitoring of live hESC-derived CMs.Methods and resultsWe used CRISPR/Cas9 to knock a mCherry reporter gene into the MYH6 locus of hESC lines, H1 and H9, enabling real-time monitoring of the generation of CMs. MYH6:mCherry+ cells express atrial or ventricular markers and display a range of cardiomyocyte action potential morphologies. At 20?days of differentiation, MYH6:mCherry+ cells show features characteristic of human CMs and can be used successfully to monitor drug-induced cardiotoxicity and oleic acid-induced cardiac arrhythmia.ConclusionWe created two MYH6:mCherry hESC reporter lines and documented the application of these lines for disease modelling relevant to cardiomyocyte biology.
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Project description:Recent discovery of a method for derivation and culture of germline-competent rat pluripotent stem cells (PSCs) enables generation of transgenic rats or knock-out rats via genetic modification of such PSCs. This opens the way to use rats, as is routine in mice, for analyses of gene functions or physiological features. In mouse or human, one widely used technique to express a gene of interest stably and ubiquitously is to insert that gene into the Rosa26 locus via gene targeting of PSCs. Rosa26 knock-in mice conditionally expressing a reporter or a toxin gene have contributed to tracing or ablation of specific cell lineages. We successfully identified a rat orthologue of the mouse Rosa26 locus. Insertion of tdTomato, a variant of red fluorescent protein, into the Rosa26 locus of PSCs of various rat strains allows ubiquitous expression of tdTomato. Through germline transmission of one Rosa26-tdTomato knock-in embryonic stem cell line, we also obtained tdTomato knock-in rats. These expressed tdTomato ubiquitously throughout their bodies, which indicates that the rat Rosa26 locus conserves functions of its orthologues in mouse and human. The new tools described here (targeting vectors, knock-in PSCs, and rats) should be useful for a variety of research using rats.
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