scholarly journals Generation and validation of versatile inducible CRISPRi embryonic stem cell and mouse model

PLoS Biology ◽  
2020 ◽  
Vol 18 (11) ◽  
pp. e3000749
Author(s):  
Rui Li ◽  
Xianyou Xia ◽  
Xing Wang ◽  
Xiaoyu Sun ◽  
Zhongye Dai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mouse Model ◽  
Ex Vivo ◽  
Embryonic Stem ◽  
Regulatory Elements ◽  
Loss Of Function ◽  
Transgenic Expression ◽  
Guide Rnas ◽  
High Throughput Screens

Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) 9 has been widely used far beyond genome editing. Fusions of deactivated Cas9 (dCas9) to transcription effectors enable interrogation of the epigenome and controlling of gene expression. However, the large transgene size of dCas9-fusion hinders its applications especially in somatic tissues. Here, we develop a robust CRISPR interference (CRISPRi) system by transgenic expression of doxycycline (Dox) inducible dCas9-KRAB in mouse embryonic stem cells (iKRAB ESC). After introduction of specific single-guide RNAs (sgRNAs), the induced dCas9-KRAB efficiently maintains gene inactivation, although it modestly down-regulates the expression of active genes. The proper timing of Dox addition during cell differentiation or reprogramming allows us to study or screen spatiotemporally activated promoters or enhancers and thereby the gene functions. Furthermore, taking the ESC for blastocyst injection, we generate an iKRAB knock-in (KI) mouse model that enables the shutdown of gene expression and loss-of-function (LOF) studies ex vivo and in vivo by a simple transduction of gRNAs. Thus, our inducible CRISPRi ESC line and KI mouse provide versatile and convenient platforms for functional interrogation and high-throughput screens of specific genes and potential regulatory elements in the setting of development or diseases.

scholarly journals Generation and Validation of Versatile Inducible CRISPRi Embryonic Stem Cell and Mouse Models

2020 ◽  
Author(s):  
Rui Li ◽  
Xianyou Xia ◽  
Xing Wang ◽  
Xiaoyu Sun ◽  
Zhongye Dai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ex Vivo ◽  
Embryonic Stem ◽  
Regulatory Elements ◽  
Loss Of Function ◽  
Transgenic Expression ◽  
Somatic Tissues ◽  
Gene Functions ◽  
High Throughput Screens

ABSTRACTCRISPR-Cas9 has been widely used far beyond genome editing. Fusions of deactivated Cas9 (dCas9) to transcription effectors enable interrogation of the epigenome and controlling of gene expression. However the large transgene size of dCas9-fusion hinders its applications especially in somatic tissues. Here, we develop a robust CRISPR interference (CRISPRi) system by transgenic expression of doxycycline (Dox) inducible dCas9-KRAB in mouse embryonic stem cells (iKRAB ESC). After introduction of specific gRNAs, the induced dCas9-KRAB efficiently maintains gene inactivation, though it exerts modest effects on active gene expression. Proper timing of Dox addition during cell differentiation or reprogramming allows us to study or screen spatiotemporally activated promoters or enhancers and thereby the gene functions. Furthermore, taking the ESC for blastocyst injection, we generate an iKRAB knockin (KI) mouse model that enables shut-down of gene expression and loss-of-function studies ex vivo and in vivo by a simple transduction of gRNAs. Thus, our inducible CRISPRi ESC line and KI mouse provide versatile and convenient platforms for functional interrogation and high-throughput screens of specific genes and potential regulatory elements in the setting of development or diseases.

scholarly journals The role of Cdx2 as a lineage specific transcriptional repressor for pluripotent network during trophectoderm and inner cell mass specification

2017 ◽  
Author(s):  
Daosheng Huang ◽  
Xiaoping Han ◽  
Ping Yuan ◽  
Amy Ralston ◽  
Lingang Sun ◽  
...  
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Regulatory Elements ◽  
Cell System ◽  
Early Embryo ◽  
General Mechanism ◽  
Mouse Development

SUMMARYThe first cellular differentiation event in mouse development leads to the formation of the blastocyst consisting of the inner cell mass (ICM) and an outer functional epithelium called trophectoderm (TE). The lineage specific transcription factor CDX2 is required for proper TE specification, where it promotes expression of TE genes, and represses expression of Pou5f1 (OCT4) by inhibiting OCT4 from promoting its own expression. However its downstream network in the developing early embryo is not fully characterized. Here, we performed high-throughput single embryo qPCR analysis in Cdx2 null embryos to identify components of the CDX2-regulated network in vivo. To identify genes likely to be regulated by CDX2 directly, we performed CDX2 ChIP-Seq on trophoblast stem (TS) cells, derived from the TE. In addition, we examined the dynamics of gene expression changes using an inducible CDX2 embryonic stem (ES) cell system, so that we could predict which CDX2-bound genes are activated or repressed by CDX2 binding. By integrating these data with observations of chromatin modifications, we were able to identify novel regulatory elements that are likely to repress gene expression in a lineage-specific manner. Interestingly, we found CDX2 binding sites within regulatory elements of key pluripotent genes such as Pou5f1 and Nanog, pointing to the existence of a novel mechanism by which CDX2 maintains repression of OCT4 in trophoblast. Our study proposes a general mechanism in regulating lineage segregation during mammalian development.

scholarly journals The Firre locus produces a trans-acting RNA molecule that functions in hematopoiesis

2019 ◽  
Author(s):  
Jordan P. Lewandowski ◽  
James C. Lee ◽  
Taeyoung Hwang ◽  
Hongjae Sunwoo ◽  
Jill M. Goldstein ◽  
...  
Keyword(s):  
Gene Expression ◽  
X Chromosome ◽  
Sex Chromosome ◽  
Noncoding Rnas ◽  
Regulatory Elements ◽  
Genetic Evidence ◽  
Loss Of Function ◽  
Lncrna Transcript ◽  
Dna And Rna

ABSTRACTRNA has been classically known to play central roles in biology, including maintaining telomeres1, protein synthesis2, and in sex chromosome compensation in certain species3,4. At the center of these important biological systems are noncoding RNAs. While thousands of long noncoding RNAs (lncRNAs) have been identified in mammalian genomes5–8, attributing RNA-based roles to lncRNA loci requires an assessment of whether the observed effect could be due to DNA regulatory elements, the act of transcription, or the lncRNA transcript. Here, we use the syntenically conserved lncRNA locus, Functional intergenic repeating RNA element (Firre), that is located on the X chromosome as a model to discriminate between DNA- and RNA-mediated effects in vivo. To this end, we generated genetically defined loss-of-function, gain-of-function, and rescue mouse models for Firre and provide genetic evidence that the Firre locus produces a trans-acting RNA. We report that: (i) Firre mutant mice have cell-specific defects during hematopoiesis and changes in gene expression that can be rescued by induction of Firre RNA from a transgene in the Firre knockout background, (ii) mice overexpressing Firre from a transgene exhibit increased levels of pro-inflammatory cytokines and impaired survival upon exposure to lipopolysaccharide, and (iii) deletion of the Firre locus did not result in changes in local gene expression on the X chromosome in 9 different biological contexts, suggesting that Firre does not function by cis-acting RNA or DNA elements. Together, our results provide genetic evidence that the Firre locus produces a trans-acting lncRNA that has physiological roles in hematopoiesis and immune function.

scholarly journals ZMYND11-MBTD1 induces leukemogenesis through hijacking NuA4/TIP60 acetyltransferase complex and a PWWP-mediated chromatin association mechanism

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jie Li ◽  
Phillip M. Galbo ◽  
Weida Gong ◽  
Aaron J. Storey ◽  
Yi-Hsuan Tsai ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Fusion Gene ◽  
Ex Vivo ◽  
Chromosomal Translocation ◽  
Regulatory Elements ◽  
Hematopoietic Stem ◽  
Its Gene ◽  
Human Acute Myeloid Leukemia ◽  
Bromodomain Proteins

AbstractRecurring chromosomal translocation t(10;17)(p15;q21) present in a subset of human acute myeloid leukemia (AML) patients creates an aberrant fusion gene termed ZMYND11-MBTD1 (ZM); however, its function remains undetermined. Here, we show that ZM confers primary murine hematopoietic stem/progenitor cells indefinite self-renewal capability ex vivo and causes AML in vivo. Genomics profilings reveal that ZM directly binds to and maintains high expression of pro-leukemic genes including Hoxa, Meis1, Myb, Myc and Sox4. Mechanistically, ZM recruits the NuA4/Tip60 histone acetyltransferase complex to cis-regulatory elements, sustaining an active chromatin state enriched in histone acetylation and devoid of repressive histone marks. Systematic mutagenesis of ZM demonstrates essential requirements of Tip60 interaction and an H3K36me3-binding PWWP (Pro-Trp-Trp-Pro) domain for oncogenesis. Inhibitor of histone acetylation-‘reading’ bromodomain proteins, which act downstream of ZM, is efficacious in treating ZM-induced AML. Collectively, this study demonstrates AML-causing effects of ZM, examines its gene-regulatory roles, and reports an attractive mechanism-guided therapeutic strategy.

scholarly journals The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1;85(7):1983]

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 319-329 ◽  
Author(s):  
S Dziennis ◽  
RA Van Etten ◽  
HL Pahl ◽  
DL Morris ◽  
TL Rothstein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Transgene Expression ◽  
Heterologous Gene Expression ◽  
Myeloid Cells ◽  
Reporter Genes ◽  
Regulatory Elements ◽  
Specific Expression ◽  
High Level

Abstract CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type- specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.

Abstract 290: Haemogenic Endocardium Contribute To Definitive Hematopoiesis During Cardiogenesis

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Haruko Nakano ◽  
Xiaoqian Liu ◽  
Armin Arshi ◽  
Ben van Handel ◽  
Rajkumar Sasidharan ◽  
...  
Keyword(s):  
De Novo ◽  
Ex Vivo ◽  
Embryonic Stem ◽  
Circulatory System ◽  
Lineage Tracing ◽  
Mouse Heart ◽  
Heart Tube ◽  
Cardiac Progenitors ◽  
Definitive Hematopoiesis

The circulatory system is the first functional organ system that develops during mammalian life. Accumulating evidences suggest that cardiac and endocardial cells can arise from a single common progenitor cell during mammalian cardiogenesis. Notably, these early cardiac progenitors express multiple hematopoietic transcription factors, consistent with previous reports. Indeed, a close relationship among cardiac, endocardial and hematopoietic lineages has been suggested in fly, zebrafish, and embryonic stem cell in vitro differentiation models. However, it is unclear when, where and how this hematopoietic gene program is in operation during in vivo mammalian cardiogenesis. Hematopoietic colony assay suggests that mouse heart explants generate myeloids and erythroids in the absence of circulation, suggesting that the heart tube is a de novo site for the definitive hematopoiesis. Lineage tracing revealed that putative cardiac-derived Nkx2-5+/Isl1+ endocardial cells give rise to CD41+ hematopoietic progenitors that contribute to definitive hematopoiesis in vivo and ex vivo during embryogenesis earlier than in the AGM region. Furthermore, Nkx2-5 and Isl1 are both required for the hemogenic activity of the endocardium. Together, identification of Nkx2-5/Isl1-dependent hemogenic endocardial cells (1) adds hematopoietic component in the cardiogenesis lineage tree, (2) changes the long-held dogma that AGM is the only major source of definitive hematopoiesis in the embryo proper, and (3) represents phylogenetically conserved fundamental mechanism of cardio-vasculo-hematopoietic differentiation pathway during the development of circulatory system.

A novel mouse model based on intersectional genetics enables unambiguous in vivo discrimination between plasmacytoid and other dendritic cells and their comparative characterization

2022 ◽  
Author(s):  
Michael Valente ◽  
Nils Collinet ◽  
Thien-Phong Vu Manh ◽  
Karima Naciri ◽  
Gilles Bessou ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Steady State ◽  
Mouse Model ◽  
Single Cell ◽  
Ex Vivo ◽  
High Specificity ◽  
Type I ◽  
Specific Expression ◽  
Physiological Functions

Plasmacytoid dendritic cells (pDC) were identified about 20 years ago, based on their unique ability to rapidly produce copious amounts of all subsets of type I and type III interferon (IFN-I/III) upon virus sensing, while being refractory to infection. Yet, the identity and physiological functions of pDC are still a matter of debate, in a large part due to their lack of specific expression of any single cell surface marker or gene that would allow to track them in tissues and to target them in vivo with high specificity and penetrance. Indeed, recent studies showed that previous methods that were used to identify or deplete pDC also targeted other cell types, including pDC-like cells and transitional DC (tDC) that were proposed to be responsible for all the antigen presentation ability previously attributed to steady state pDC. Hence, improving our understanding of the nature and in vivo choreography of pDC physiological functions requires the development of novel tools to unambiguously identify and track these cells, including in comparison to pDC-like cells and tDC. Here, we report successful generation of a pDC-reporter mouse model, by using an intersectional genetic strategy based on the unique co-expression of Siglech and Pacsin1 in pDC. This pDC-Tomato mouse strain allows specific ex vivo and in situ detection of pDC. Breeding them with Zbtb46GFP mice allowed side-by-side purification and transcriptional profiling by single cell RNA sequencing of bona fide pDC, pDC-like cells and tDC, in comparison to type 1 and 2 conventional DC (cDC1 and cDC2), both at steady state and during a viral infection, revealing diverging activation patterns of pDC-like cells and tDC. Finally, by breeding pDC-Tomato mice with Ifnb1EYFP mice, we determined the choreography of pDC recruitment to the micro-anatomical sites of viral replication in the spleen, with initially similar but later divergent behaviors of the pDC that engaged or not into IFN-I production. Our novel pDC-Tomato mouse model, and newly identified gene modules specific to combinations of DC types and activations states, will constitute valuable resources for a deeper understanding of the functional division of labor between DC types and its molecular regulation at homeostasis and during viral infections.

scholarly journals Evolutionary Analysis of Transcriptional Regulation Mediated by Cdx2 in Rodents

2021 ◽  
Author(s):  
Weizheng Liang ◽  
Guipeng Li ◽  
Huanhuan Cui ◽  
Yukai Wang ◽  
Wencheng Wei ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Amino Acid ◽  
Dna Binding ◽  
Phenotypic Diversity ◽  
Embryonic Stem ◽  
Regulatory Elements ◽  
Evolutionary Analysis ◽  
Protein Sequence Analysis ◽  
Systematic Analysis

AbstractDifferences in gene expression, which can arise from divergence in cis-regulatory elements or alterations in transcription factors binding specificity, are one of the most important causes of phenotypic diversity during evolution. By protein sequence analysis, we observed high sequence conservation in the DNA binding domain (DBD) of the transcription factor Cdx2 across many vertebrates, whereas three amino acid changes were exclusively found in mouse Cdx2 (mCdx2), suggesting potential positive selection in the mouse lineage. Multi-omics analyses were then carried out to investigate the effects of these changes. Surprisingly, there were no significant functional differences between mCdx2 and its rat homologue (rCdx2), and none of the three amino acid changes had any impact on its function. Finally, we used rat-mouse allodiploid embryonic stem cells (RMES) to study the cis effects of Cdx2-mediated gene regulation between the two rodents. Interestingly, whereas Cdx2 binding is largely divergent between mouse and rat, the transcriptional effect induced by Cdx2 is conserved to a much larger extent.Author summaryOur study 1) represented a first systematic analysis of species-specific adaptation in DNA binding pattern of transcription factor. Although the mouse-specific amino acid changes did not manifest functional impact in our system, several explanations may account for it (See Discussion part for the detail); 2) represented a first study of cis-regulation between two reproductively isolated species by using a novel allodiploid system; 3) demonstrated a higher conservation of transcriptional output than that of DNA binding, suggesting the evolvability/plasticity of the latter; 4) finally provided a rich data resource for Cdx2 mediated regulation, including gene expression, chromatin accessibility and DNA binding etc.

scholarly journals Healthy versus inflamed lung environments differentially effect MSCs

2021 ◽  
pp. 2004149
Author(s):  
Sara Rolandsson Enes ◽  
Thomas H. Hampton ◽  
Jayita Barua ◽  
David H. McKenna ◽  
Claudia C. dos Santos ◽  
...  
Keyword(s):  
Gene Expression ◽  
Healthy Volunteers ◽  
Cell Injury ◽  
Ex Vivo ◽  
Lavage Fluid ◽  
Cell Therapies ◽  
Inflammatory Microenvironment ◽  
Self Recognition ◽  
Clinical Investigations

BackgroundDespite increased interest in MSC-based cell therapies for the acute respiratory distress syndrome (ARDS), clinical investigations have not yet been successful and understanding of the potential in vivo mechanisms of MSC actions in ARDS remain limited. ARDS is driven by an acute severe innate immune dysregulation, often characterised by inflammation, coagulation, and cell injury. How this inflammatory microenvironment influences MSC functions remains to be determined.AimTo comparatively assess how the inflammatory environment present in ARDS lungs versus the lung environment present in healthy volunteers alters MSC behaviors.MethodsClinical grade human bone marrow-derived MSCs (hMSCs) were exposed to bronchoalveolar lavage fluid (BALF) samples obtained from ARDS patients or from healthy volunteers. Following exposure, hMSCs and their conditioned media were evaluated for a broad panel of relevant properties including viability, levels of expression of inflammatory cytokines, gene expression, cell surface HLA expression, and activation of coagulation and complement pathways.ResultsPro-inflammatory, pro-coagulant, and major histocompatibility complex (self recognition) related gene expression was markedly up-regulated in hMSCs exposed ex vivo to BALF obtained from healthy volunteers. In contrast, these changes were less apparent and often opposite in hMSCs exposed to ARDS BALF samples.ConclusionThese data provide new insights into how hMSCs behave in healthy versus inflamed lung environments strongly suggesting that the inflamed environment in ARDS induces hMSC responses potentially benefical for cell survival and actions. This further highlights the need to understand how different disease environments affect hMSC functions.

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