Developmentally regulated gene expression of thrombomodulin in postimplantation mouse embryos

Development ◽  
1996 ◽  
Vol 122 (7) ◽  
pp. 2271-2281 ◽  
Author(s):  
H. Weiler-Guettler ◽  
W.C. Aird ◽  
H. Rayburn ◽  
M. Husain ◽  
R.D. Rosenberg
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Reporter Gene ◽  
Es Cells ◽  
Embryonic Stem ◽  
Yolk Sac ◽  
Regulatory Control ◽  
Specific Gene ◽  
Anatomical Site ◽  
Regulated Gene Expression

Embryonic lethality of thrombomodulin-deficient mice has indicated an essential role for this regulator of blood coagulation in murine development. Here, the embryonic expression pattern of thrombomodulin was defined by surveying beta-galactosidase activity in a mouse strain in which the reporter gene was placed under the regulatory control of the endogenous thrombomodulin promoter via homologous recombination in embryonic stem cells. The murine trophoblast was identified as a previously unrecognized anatomical site where TM expression is conserved between humans and mice and may exert a critical function during postimplantation development. Targeted reporter gene expression in mesodermal precursors of the endothelial cell lineage defined thrombomodulin as an early marker of vascular differentiation. Analysis of the thrombomodulin promoter in differentiating ES cells and in transgenic mice provided evidence for a disparate and cell type-specific gene regulatory control mechanism in the parietal yolk sac. The thrombomodulin promoter as defined in this study will allow the targeting of gene expression to the parietal yolk sac of transgenic mice and the initiation of investigations into the role of parietal endoderm in placental function.

Murine gastrulation requires HNF-4 regulated gene expression in the visceral endoderm: tetraploid rescue of Hnf-4(−/−) embryos

Development ◽  
1997 ◽  
Vol 124 (2) ◽  
pp. 279-287 ◽  
Author(s):  
S.A. Duncan ◽  
A. Nagy ◽  
W. Chan
Keyword(s):  
Gene Expression ◽  
Genetic Manipulation ◽  
Es Cells ◽  
Critical Role ◽  
Embryonic Stem ◽  
Retinol Binding Protein ◽  
Specific Gene ◽  
Visceral Endoderm ◽  
Regulated Gene Expression

Immediately prior to gastrulation the murine embryo consists of an outer layer of visceral endoderm (VE) and an inner layer of ectoderm. Differentiation and migration of the ectoderm then occurs to produce the three germ layers (ectoderm, embryonic endoderm and mesoderm) from which the fetus is derived. An indication that the VE might have a critical role in this process emerged from studies of Hnf-4(−/−) mouse embryos which fail to undergo normal gastrulation. Since expression of the transcription factor HNF-4 is restricted to the VE during this phase of development, we proposed that HNF-4-regulated gene expression in the VE creates an environment capable of supporting gastrulation. To address this directly we have exploited the versatility of embryonic stem (ES) cells which are amenable to genetic manipulation and can be induced to form VE in vitro. Moreover, embryos derived solely from ES cells can be generated by aggregation with tetraploid morulae. Using Hnf-4(−/−) ES cells we demonstrate that HNF-4 is a key regulator of tissue-specific gene expression in the VE, required for normal expression of secreted factors including alphafetoprotein, apolipoproteins, transthyretin, retinol binding protein, and transferrin. Furthermore, specific complementation of Hnf-4(−/−) embryos with tetraploid-derived Hnf-4(+/+) VE rescues their early developmental arrest, showing conclusively that a functional VE is mandatory for gastrulation.

scholarly journals Non-invasive somatotransgenic bioimaging in living animals

F1000Research ◽  
2020 ◽  
Vol 9 ◽  
pp. 1216 ◽  
Author(s):  
Juliette M. Delhove ◽  
Rajvinder Karda ◽  
Lorna M. FitzPatrick ◽  
Suzanne M.K. Buckley ◽  
Simon N. Waddington ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Viral Vectors ◽  
Es Cells ◽  
Embryonic Stem ◽  
Whole Body ◽  
Luciferase Reporter ◽  
Gene Promoters ◽  
Luciferase Reporter Gene ◽  
Endogenous Gene

Bioluminescence imaging enables noninvasive quantification of luciferase reporter gene expression in transgenic tissues of living rodents. Luciferase transgene expression can be regulated by endogenous gene promoters after targeted knock-in of the reporter gene, usually within the first intron of the gene. Even using CRISPR/Cas9 mediated genome editing this can be a time consuming and costly process. The generation of germline transgenic (GLT) rodents by targeted genomic integration of a gene expression cassette in embryonic stem (ES) cells is commonplace but results in the wastage of large numbers of animals during colony generation, back-crossing and maintenance. Using a synthetic/truncated promoter-driven luciferase gene to study promoter activity in a given tissue or organ of a GLT also often results in unwanted background luciferase activity during whole-body bioluminescent imaging as every cell contains the reporter. We have developed somatotransgenic bioimaging; a method to generate tissue-restricted transcription factor activated luciferase reporter (TFAR) cassettes in rodents that substantially reduces the number of animals required for experimentation. Bespoke designed TFARs are delivered to newborn pups using viral vectors targeted to specific organs by tissue-tropic pseudotypes. Retention and proliferation of TFARs is facilitated by stem/progenitor cell transduction and immune tolerance to luciferase due to the naïve neonatal immune system. We have successfully applied both lentiviral and adeno-associated virus (AAV) vectors in longitudinal rodent studies, targeting TFARs to the liver and brain during normal development and in well-established disease models. Development of somatotransgenic animals has broad applicability to non-invasively determine mechanistic insights into homeostatic and disease states and assess toxicology and efficacy testing. Somatotransgenic bioimaging technology is superior to current whole-body, light-emitting transgenic models as it reduces the numbers of animals used by generating only the required number of animals. It is also a refinement over current technologies given the ability to use conscious, unrestrained animals.

500. THE MANIPULATION OF THE EPIGENETIC MARK HISTONE 3 LYSINE 9 TRIMETHYLATION IN DONOR CELLS AND ITS EFFECTS ON THE DEVELOPMENT OF CLONED MOUSE EMBRYOS

2009 ◽  
Vol 21 (9) ◽  
pp. 101
Author(s):  
J. Antony ◽  
F. Oback ◽  
R. Broadhurst ◽  
S. Cole ◽  
C. Graham ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nuclear Transfer ◽  
Expression Profiles ◽  
Es Cells ◽  
Embryonic Stem ◽  
Histone Demethylase ◽  
Specific Gene ◽  
Epigenetic Mark ◽  
Epigenetic Marks ◽  
Donor Cells

To produce live cloned mammals from adult somatic cells the nuclei of these cells must be first reprogrammed from a very restricted, cell lineage-specific gene expression profile to an embryo-like expression pattern, compatible with embryonic development. Although this has been achieved in a number of species the efficiency of cloning remains very low. Inadequate reprogramming of epigenetic marks in the donor cells correlated with aberrant embryonic gene expression profiles has been identified as a key cause of this inefficiency. Some of the most common epigenetic marks are chemical modifications of histones, the main structural proteins of chromatin. A range of different histone modifications, including acetylation and methylation, exists and can be attributed to either repression or activation of genes. One epigenetic mark which is known to be very stable and difficult to remove during reprogramming is the trimethylation of lysine 9 in histone H3 (H3K9Me3). To test the hypothesis that H3K9Me3 marks are a major stumbling block for successful cloning we are attempting to remove these marks by overexpression of the H3K9Me3 specific histone demethylase, jmjd2b, in donor cells, prior to their use for nuclear transfer. We have engineered mouse embryonic stem (ES) cells for the tet inducible expression of a fusion protein with a functional jmjd2b or non-functional mutant jmjd2b histone demethylase. Approximately 94% and 88% of the cells can be induced for the expression of functional and mutant jmjd2b-EGFP in the respective ES cell lines. Immunofluorescence analyses have shown that induction of functional jmjd2b-EGFP results in an approximately 50% reduction of H3K9Me3 levels compared to non-induced cells and induced mutant jmjd2b-EGFP cells. The comparison of the in-vitro embryo development following nuclear transfer with induced and non-induced donor cells show significantly better overall development to blastocysts and morulae from induced donor cells with reduced H3K9Me3 levels.

A large-scale gene-trap screen for insertional mutations in developmentally regulated genes in mice.

Genetics ◽  
1995 ◽  
Vol 139 (2) ◽  
pp. 889-899 ◽  
Author(s):  
W Wurst ◽  
J Rossant ◽  
V Prideaux ◽  
M Kownacka ◽  
A Joyner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Large Scale ◽  
Es Cells ◽  
Embryonic Stem ◽  
Gene Trap ◽  
Reporter Gene Expression ◽  
Chimeric Mouse ◽  
Developmentally Regulated ◽  
Active Transcription

Abstract We have used a gene-trap vector and mouse embryonic stem (ES) cells to screen for insertional mutations in genes developmentally regulated at 8.5 days of embryogenesis (dpc). From 38,730 cell lines with vector insertions, 393 clonal integrations had disrupted active transcription units, as assayed by beta-galactosidase reporter gene expression. From these lines, 290 clones were recovered and injected into blastocysts to assay for reporter gene expression in 8.5-dpc chimeric mouse embryos. Of these, 279 clones provided a sufficient number of chimeric embryos for analysis. Thirty-six (13%) showed restricted patterns of reporter-gene expression, 88 (32%) showed widespread expression and 155 (55%) failed to show detectable levels of expression. Further analysis showed that approximately one-third of the clones that did not express detectable levels of the reporter gene at 8.5 dpc displayed reporter gene activity at 12.5 dpc. Thus, a large proportion of the genes that are expressed in ES cells are either temporally or spatially regulated during embryogenesis. These results indicate that gene-trap mutageneses in embryonic stem cells provide an effective approach for isolating mutations in a large number of developmentally regulated genes.

scholarly journals Octamer and Sox Elements Are Required for Transcriptional cis Regulation of Nanog Gene Expression

2005 ◽  
Vol 25 (6) ◽  
pp. 2475-2485 ◽  
Author(s):  
Takao Kuroda ◽  
Masako Tada ◽  
Hiroshi Kubota ◽  
Hironobu Kimura ◽  
Shin-ya Hatano ◽  
...  
Keyword(s):  
Gene Expression ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Es Cells ◽  
Embryonic Stem ◽  
Specific Gene ◽  
Mobility Shift ◽  
Adjacent Pair ◽  
Embryonic Germ Cells ◽  
Cell Extracts

ABSTRACT The pluripotential cell-specific gene Nanog encodes a homeodomain-bearing transcription factor required for maintaining the undifferentiated state of stem cells. However, the molecular mechanisms that regulate Nanog gene expression are largely unknown. To address this important issue, we used luciferase assays to monitor the relative activities of deletion fragments from the 5′-flanking region of the gene. An adjacent pair of highly conserved Octamer- and Sox-binding sites was found to be essential for activating pluripotential state-specific gene expression. Furthermore, the 5′-end fragment encompassing the Octamer/Sox element was sufficient for inducing the proper expression of a green fluorescent protein reporter gene even in human embryonic stem (ES) cells. The potential of OCT4 and SOX2 to bind to this element was verified by electrophoretic mobility shift assays with extracts from F9 embryonal carcinoma cells and embryonic germ cells derived from embryonic day 12.5 embryos. However, in ES cell extracts, a complex of OCT4 with an undefined factor preferentially bound to the Octamer/Sox element. Thus, Nanog transcription may be regulated through an interaction between Oct4 and Sox2 or a novel pluripotential cell-specific Sox element-binding factor which is prominent in ES cells.

scholarly journals The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Yick W Fong ◽  
Jaclyn J Ho ◽  
Carla Inouye ◽  
Robert Tjian
Keyword(s):  
Stem Cell ◽  
Es Cells ◽  
Embryonic Stem ◽  
In Vitro Transcription ◽  
Specific Gene ◽  
Transcriptional Level ◽  
Ips Cell ◽  
Ribonucleoprotein Complex ◽  
Transcription System

Acquisition of pluripotency is driven largely at the transcriptional level by activators OCT4, SOX2, and NANOG that must in turn cooperate with diverse coactivators to execute stem cell-specific gene expression programs. Using a biochemically defined in vitro transcription system that mediates OCT4/SOX2 and coactivator-dependent transcription of the Nanog gene, we report the purification and identification of the dyskerin (DKC1) ribonucleoprotein complex as an OCT4/SOX2 coactivator whose activity appears to be modulated by a subset of associated small nucleolar RNAs (snoRNAs). The DKC1 complex occupies enhancers and regulates the expression of key pluripotency genes critical for self-renewal in embryonic stem (ES) cells. Depletion of DKC1 in fibroblasts significantly decreased the efficiency of induced pluripotent stem (iPS) cell generation. This study thus reveals an unanticipated transcriptional role of the DKC1 complex in stem cell maintenance and somatic cell reprogramming.

The BMP/BMPR/Smad pathway directs expression of the erythroid-specific EKLF and GATA1 transcription factors during embryoid body differentiation in serum-free media

Development ◽  
2002 ◽  
Vol 129 (2) ◽  
pp. 539-549 ◽  
Author(s):  
Carrie A. Adelman ◽  
Subrata Chattopadhyay ◽  
James J. Bieker
Keyword(s):  
Embryoid Body ◽  
Es Cells ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Dominant Negative ◽  
Erythroid Cell ◽  
Specific Gene ◽  
Smad Pathway ◽  
Cellular Expression ◽  
Novel Approach

Erythroid cell-specific gene regulation during terminal differentiation is controlled by transcriptional regulators, such as EKLF and GATA1, that themselves exhibit tissue-restricted expression patterns. Their early expression, already in evidence within multipotential hematopoietic cell lines, has made it difficult to determine what extracellular effectors and transduction mechanisms might be directing the onset of their own transcription during embryogenesis. To circumvent this problem, we have taken the novel approach of investigating whether the ability of embryonic stem (ES) cells to mimic early developmental patterns of cellular expression during embryoid body (EB) differentiation can address this issue. We first established conditions whereby EBs could form efficiently in the absence of serum. Surprisingly, in addition to mesoderm, these cells expressed hemangioblast and hematopoietic markers. However, they did not express the committed erythroid markers EKLF and GATA1, nor the terminally differentiated β-like globin markers. Using this system, we determined that EB differentiation in BMP4 was necessary and sufficient to recover EKLF and GATA1 expression and could be further stimulated by the inclusion of VEGF, SCF, erythropoietin and thyroid hormone. EBs were competent to respond to BMP4 only until day 4 of differentiation, which coincides with the normal onset of EKLF expression. The direct involvement of the BMP/Smad pathway in this induction process was further verified by showing that erythroid expression of a dominant negative BMP1B receptor or of the inhibitory Smad6 protein prevented induction of EKLF or GATA1 even in the presence of serum. Although Smad1, Smad5 and Smad8 are all expressed in the EBs, BMP4 induction of EKLF and GATA1 transcription is not immediate. These data implicate the BMP/Smad induction system as being a crucial pathway to direct the onset of EKLF and GATA1 expression during hematopoietic differentiation and demonstrate that EB differentiation can be manipulated to study induction of specific genes that are expressed early within a lineage.

The responsiveness of embryonic stem cells to alpha and beta interferons provides the basis of an inducible expression system for analysis of developmental control genes

1993 ◽  
Vol 13 (12) ◽  
pp. 7971-7976
Author(s):  
L M Whyatt ◽  
A Düwel ◽  
A G Smith ◽  
P D Rathjen
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Expression System ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Expression Vectors ◽  
Developmental Control ◽  
Developmental Potential

Embryonic stem (ES) cells, derived from the inner cell mass of the preimplantation mouse embryo, are used increasingly as an experimental tool for the investigation of early mammalian development. The differentiation of these cells in vitro can be used as an assay for factors that regulate early developmental decisions in the embryo, while the effects of altered gene expression during early embryogenesis can be analyzed in chimeric mice generated from modified ES cells. The experimental versatility of ES cells would be significantly increased by the development of systems which allow precise control of heterologous gene expression. In this paper, we report that ES cells are responsive to alpha and beta interferons (IFNs). This property has been exploited for the development of inducible ES cell expression vectors, using the promoter of the human IFN-inducible gene, 6-16. The properties of these vectors have been analyzed in both transiently and stably transfected ES cells. Expression was minimal or absent in unstimulated ES cells, could be stimulated up to 100-fold by treatment of the cells with IFN, and increased in linear fashion with increasing levels of IFN. High levels of induced expression were maintained for extended periods of time in the continuous presence of the inducing signal or following a 12-h pulse with IFN. Treatment of ES cells with IFN did not affect their growth or differentiation in vitro or compromise their developmental potential. This combination of features makes the 6-16-based expression vectors suitable for the functional analysis of developmental control control genes in ES cells.

scholarly journals Improved Experimental Procedures for Achieving Efficient Germ Line Transmission of Nonobese Diabetic (NOD)-Derived Embryonic Stem Cells

2004 ◽  
Vol 5 (3) ◽  
pp. 219-226 ◽  
Author(s):  
Satoko Arai ◽  
Christina Minjares ◽  
Seiho Nagafuchi ◽  
Toru Miyazaki
Keyword(s):  
High Efficiency ◽  
Germ Line ◽  
Gene Knockout ◽  
Es Cells ◽  
Embryonic Stem ◽  
Nod Mice ◽  
Insulin Dependent Diabetes Mellitus ◽  
Transmission Efficiency ◽  
Dependent Diabetes Mellitus ◽  
Specific Gene

The manipulation of a specific gene in NOD mice, the best animal model for insulin-dependent diabetes mellitus (IDDM), must allow for the precise characterization of the functional involvement of its encoded molecule in the pathogenesis of the disease. Although this has been attempted by the cross-breeding of NOD mice with many gene knockout mice originally created on the 129 or C57BL/6 strain background, the interpretation of the resulting phenotype(s) has often been confusing due to the possibility of a known or unknown disease susceptibility locus (e.g.,Iddlocus) cosegregating with the targeted gene from the diabetes-resistant strain. Therefore, it is important to generate mutant mice on a pure NOD background by using NOD-derived embryonic stem (ES) cells. By using the NOD ES cell line established by Nagafuchi and colleagues in 1999 (FEBSLett., 455, 101–104), the authors reexamined various conditions in the context of cell culture, DNA transfection, and blastocyst injection, and achieved a markedly improved transmission efficiency of these NOD ES cells into the mouse germ line. These modifications will enable gene targeting on a “pure” NOD background with high efficiency, and contribute to clarifying the physiological roles of a variety of genes in the disease course of IDDM.

scholarly journals Differences in Gene Expression between Wild Type and Hoxa1 Knockout Embryonic Stem Cells after Retinoic Acid Treatment or Leukemia Inhibitory Factor (LIF) Removal

2005 ◽  
Vol 280 (16) ◽  
pp. 16484-16498 ◽  
Author(s):  
Eduardo Martinez-Ceballos ◽  
Pierre Chambon ◽  
Lorraine J. Gudas
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Retinoic Acid ◽  
Leukemia Inhibitory Factor ◽  
Target Genes ◽  
Hox Genes ◽  
Es Cells ◽  
Embryonic Stem ◽  
Inhibitory Factor ◽  
Hoxa Cluster

Homeobox (Hox) genes encode a family of transcription factors that regulate embryonic patterning and organogenesis. In embryos, alterations of the normal pattern of Hox gene expression result in homeotic transformations and malformations. Disruption of theHoxa1gene, the most 3′ member of the Hoxa cluster and a retinoic acid (RA) direct target gene, results in abnormal ossification of the skull, hindbrain, and inner ear deficiencies, and neonatal death. We have generated Hoxa1-/-embryonic stem (ES) cells (named Hoxa1-15) from Hoxa1-/-mutant blastocysts to study the Hoxa1 signaling pathway. We have characterized in detail these Hoxa1-/-ES cells by performing microarray analyses, and by this technique we have identified a number of putative Hoxa-1 target genes, including genes involved in bone development (e.g. Col1a1,Postn/Osf2, and the bone sialoprotein gene orBSP), genes that are expressed in the developing brain (e.g. Nnat,Wnt3a,BDNF,RhoB, andGbx2), and genes involved in various cellular processes (e.g. M-RAS,Sox17,Cdkn2b,LamA1,Col4a1,Foxa2,Foxq1,Klf5, andIgf2). Cell proliferation assays and Northern blot analyses of a number of ES cell markers (e.g. Rex1,Oct3/4,Fgf4, andBmp4) suggest that the Hoxa1 protein plays a role in the inhibition of cell proliferation by RA in ES cells. Additionally, Hoxa1-/-ES cells express high levels of various endodermal markers, includingGata4andDab2, and express much lessFgf5after leukemia inhibitory factor (LIF) withdrawal. Finally, we propose a model in which the Hoxa1 protein mediates repression of endodermal differentiation while promoting expression of ectodermal and mesodermal characteristics.

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