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Friday, November 13, 2020 | History

2 edition of Identification of a novel stem cell specific candidate silencer element in retrovirus vectors. found in the catalog.

Identification of a novel stem cell specific candidate silencer element in retrovirus vectors.

Jason Chung-Sheung Ho

Identification of a novel stem cell specific candidate silencer element in retrovirus vectors.

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Published .
Written in English


About the Edition

The silencing of retrovirus vectors in stem cells poses a problem for their use in gene therapy and for studying stem cell biology. The identification of retrovirus silencer elements has led to the development of vectors with improved expression but these vectors are still susceptible to silencing in stem cells. DNase I footprinting across the entire MSCV retrovirus vector was performed, comparing F9 and NIH3T3 cell nuclear extracts. The results revealed differences in footprints and hypersensitive bases within the essential U5 and psi regions. Electrophoretic mobility shift assays were carried out to confirm binding and to identify the consensus sites through mutational analysis. Functional characterization of the U5 and psi regions using an episome repression assay identified a novel stem cell specific potential silencer element within the psi region.

The Physical Object
Pagination71 leaves.
Number of Pages71
ID Numbers
Open LibraryOL19217312M
ISBN 100494073942

Research Description. The burgeoning epidemic of obesity and type 2 diabetes mellitus presents a major health and therapeutic challenge. Transcriptional regulation is the fundamental control mechanism for metabolism, but a gap remains in our knowledge of gene regulatory pathways that control lipid and glucose homeostasis.   Multipotent mesenchymal stem cells (MSCs) can undergo self-renewal and give rise to multi-lineages under given differentiation cues. It is frequently desirable to achieve a stable and high level of transgene expression in MSCs in order to elucidate possible molecular mechanisms through which MSC self-renewal and lineage commitment are regulated. Retroviral or lentiviral vector-mediated gene. Research Description. Topic 1. Mechanisms underlying dopamine neurogenesis The floor plate, the ventral organizing center in the embryonic neural tube, patterns the neural tube by secreting the potent morphogen Shh. Using genetic fate mapping, we have recently shown that the midbrain floor plate, unlike the hindbrain and spinal cord floor plate, is neurogenic and is the source of midbrain.


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Identification of a novel stem cell specific candidate silencer element in retrovirus vectors. by Jason Chung-Sheung Ho Download PDF EPUB FB2

Evidence for a stem cell-specific repressor of Moloney murine leukemia virus expression in embryonal carcinoma cells.

Mol Cell Biol. Aug; 10 (8)– [PMC free article] Mann R, Mulligan RC, Baltimore D. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. by:   Silencing of retroviruses in the human genome is a crucial step in the production of induced pluripotent stem cells (iPSCs) from somatic cells.

Researchers led by. Embryonic stem cells have the potential to differentiate into any type of cell in the human body. Once differentiated though, the newly minted somatic cells live out the rest of their days as that. Embryonic stem cells (ESCs) and other primitive stem cells of mice have been known for more than 30 years to potently block retrovirus replication ion of ESCs by.

These silencers play an important role in transcriptional shut down of MoMLV LTR expression in ES cells. Other retroviral vectors designed to overcome stem cell silencing have deleted or mutated some of the silencers.

One example already used in iPS cell experiments is the murine stem cell virus (MSCV) derived vector (pMIG).Cited by: Retroviral vectors are silenced in embryonic stem (ES) cells by epigenetic mechanisms whose kinetics are poorly understood.

We show here that a 3′D4Z4 insulator directs retroviral expression with persistent but variable expression for up to 5 months.

Combining an internal 3′D4Z4 with HS4 insulators in the long terminal repeats (LTRs) shows that these elements cooperate, and defines the. Retrovirus vector designs for stem cells should delete virus silencer elements, incorporate strong positive regulatory elements and insulators, and avoid non-mammalian reporter genes.

In summary, we have developed a novel lentiviral vector-based method of screening candidate elements for insulator activity, and have used this method to identify two new insulator elements capable of improving the safety of retroviral vectors without diminishing vector titers or expression.

A stem cell-specific silencer in the primer-binding site of a retrovirus from novel retroviral vectors containing three modifications in the MoMuLV-based retroviral vector: presence of the. To prevent insertional mutagenesis arising from retroviral reactivation, cells of embryonic origin possess a unique capacity to silence retroviruses.

Given the distinct modes of X chromosome inactivation between embryonic and extraembryonic lineages, we investigated paradigms of viral extinction.

We show that trophectoderm stem cells do not silence retroviral transcription, whereas. Petersen R, Kempler G, Barklis E. A stem cell-specific silencer in the primer-binding site of a retrovirus.

Mol Cell Biol. Mar; 11 (3)– [PMC free article] Prince VE, Rigby PW. Derivatives of Moloney murine sarcoma virus capable of being transcribed in embryonal carcinoma stem cells have gained a functional Sp1 binding site.

These studies suggest that vectors encoding one or more genes involved in T-cell development, for example specific T-cell receptors, CD4, CD8, or interleukins, may be introduced into differ- ent target cells, such as lineage-restricted stem cells, T-cell precursors or lymphocytes, enabling their development to be examined in diverse host.

Identification of a novel stem cell specific candidate silencer element in retrovirus vectors. book The capacity to self-renew is dependent on the capacity of stem cells to retain over time a specific epigenetic identity, rather than to change or transform it.

(c) The term cancer stem cells (CSCs) is an operational definition (i.e., it is based on the experimental fulfillment of a specific set of functional properties). It identifies a. Induced pluripotent stem (iPS) cells can be generated using retroviral vectors expressing Oct4, Klf4, Sox2, and cMyc.

Most prior studies have required multiple retroviral vectors for reprogramming, resulting in high numbers of genomic integrations in iPS cells and limiting their use for therapeutic applications.

To overcome silencing, retroviral vector designs mutate or delete known silencer elements in or adjacent to the long terminal repeats (LTRs).

1, 2 However, even self-inactivating (SIN) retroviral vectors with a strong internal promoter are subject to silencing in embryonic stem (ES) cells.

3 Thus, further improvements rely on removing. Historically, many key milestones have driven progress in the field of stem cell research [Fig. 1] More than half a century ago, inthe first stem cells were described by Drs.

James A. Till and Ernest A. McCulloch at the University of Toronto in Canada [].They found that stem cells derived from mouse bone marrow cells had the ability to differentiate into a variety of cell types, and. A large number of viral vector systems, including those based on herpes simplex virus type 1 (HSV-1), retrovirus, adenovirus, and adeno-associated virus (AAV), have been developed over the past two decades.

Reducing or eliminating toxic components, while conserving the efficiency of gene transfer, has been the primary goal of these efforts. All currently available vector systems have their. Introduction Approach to Gene Transfer for Platelet Disorders Hematopoietic Stem Cells as Targets Preparation of the Recipient’s Marrow Analysis of the Correction of the Phenotype for Platelet Disorders Vectors Advances in Retrovirus-Mediated Transduction for Platelets Promoters and Locus Control Regions for Megakaryocyte-Specific.

To overcome silencing, retroviral vector designs mutate or delete known silencer elements in or adjacent to the long terminal repeats (LTRs). 1,2 However, even self-inactivating (SIN) retroviral vectors with a strong internal promoter are subject to silencing in embryonic stem (ES) cells.

3 Thus, further improvements rely on removing additional. Silencing resistant retrovirus vectors have been developed by removal of these silencer elements, and silencing in stem cells may ultimately be blocked using appropriate insulator elements.

View. Bui et al. use replication-defective and persistent Sendai virus (SeVdp)-based vectors to monitor retroviral silencing during reprogramming and find that the silencing occurs earlier than the acquisition of pluripotency.

Insertional chromatin immunoprecipitation (iChIP) identifies TAF-Iα, a SET/TAF-I isoform predominant in ESCs, as a factor that facilitates retroviral silencing.

Moloney murine leukemia virus (M-MLV) replication is restricted in embryonic carcinoma (EC) and embryonic stem (ES) cells, likely to protect the germ line from insertional mutagenesis. Proviral DNAs are potently silenced at the level of transcription in these cells.

This silencing is largely due to an unidentified trans-acting factor that is thought to bind to the primer binding site (PBS) of. Peter Pasceri's 75 research works with 1, citations and 4, reads, including: Shifts in Ribosome Engagement Impact Key Gene Sets in Neurodevelopment and Ubiquitination in Rett Syndrome.

Pairing is not specific to a certain cell cycle stage. A) Histogram of PFA fixed ES cells stained with DyeCycle Violet showing DNA content distribution with peaks for G1 and G2 phase cells. In recent years, stem cell therapy has become a very promising and advanced scientific research topic.

The development of treatment methods has evoked great expectations. This paper is a review focused on the discovery of different stem cells and the potential therapies based on these cells. The genesis of stem cells is followed by laboratory steps of controlled stem cell culturing and.

The scope related to stem cells is broad and includes topics in stem cell biology covering basic, clinical, biotechnology, regulatory, and ethical aspects of embryonic and adult stem cells, induced pluripotent stem cells, fate choice, specific lineage and cloning issues, translational studies, and diseases where stem cell anomalies play a key.

Negative regulation of cytokine gene transcription is an important mechanism in maintaining homeostasis of immune function. In this study, we characterized a silencer element in the human interleukin-3 gene promoter that is responsible for the cell-specific expression of interleukin This silencer activity was proposed to be mediated by an unidentified nuclear inhibitory protein (NIP).

Vectors have been modified to include elements, which increase infection of specific cell types and allow retention of transgene sequences, either as replicating episomal elements or through site-specific integration into the cell genome, and provide the ability to control transgene expression.

Introduction. Gammaretroviral vectors derived from Moloney murine leukemia virus (MLV) integrate into the host cell genome and therefore have been widely used for stable gene transfer ().One of the main limitations of gammaretroviral vectors, however, is their dependence on nuclear membrane dissolution during mitosis for access to and integration into target cell chromosomes ().

The authors conclude that a similar population of SP stem cells can be derived from either adult mouse bone marrow or skeletal muscle, and suggest "there may be some direct relationship between bone marrow-derived stem cells and other tissue- or organ-specific cells".

Thus, stem cell or progenitor cell types from various mesodermally-derived. Stem cells have been the object of much excitement and controversy amongst both scientists and the general population.

Surprisingly, though, not everybody understands the basic properties of stem cells, let alone the fact that there is more than one type of cell that falls within the “stem cell” category.

Here, I’ll lay out the basic concepts of stem cell biology as a background for. A retrovirus called HERV-H, which inserted itself into the human genome millions of years ago, may play an important role in pluripotent stem cells.

The. The availability of neuronal and astrocyte-targeting vectors will allow dissociation of cell autonomous and cell nonautonomous functions of key gene products in vivo. Similar tissue and cell-specific patterns can be achieved in stem cells using cell/tissue-specific promoters and miRNA [43, 79, 86,].

The chromatin insulator cHS4 can reduce silencing chromosomal position effects and genotoxicity associated with integrating viral vectors. However, the fully active version of this element can also reduce vector titers and is only partially effective.

In order to identify alternatives to cHS4, we developed a functional lentiviral vector-based reporter screen for enhancer-blocking insulators. Stem cells are cells that can differentiate into other types of cells, and can also divide in self-renewal to produce more of the same type of stem cells.

Telomerase is active in normal stem cells. In mammals, there are two broad types of stem cells: embryonic stem cells, which are isolated from the inner cell mass of blastocysts in early embryonic development, and adult stem cells, which.

Results. Exon 1 of Human β3Gal-T5 Is Derived from an Endogenous Retroviral LTR. A screen to identify genes containing HERVs within their UTRs revealed that the first exon of the β3Gal-T5 gene is derived from a HERV ().The HERV element is a bp solitary LTR, type MLT2B3, of the ERV-L family and is in the same orientation as the gene.

The generation of induced pluripotent stem cells (iPSCs) has opened up a new scientific frontier in medicine. This technology has made it possible to obtain pluripotent stem cells from individuals with genetic disorders.

Because iPSCs carry the identical genetic anomalies related to those disorders, iPSCs are an ideal platform for medical research. Stem Cell Research is dedicated to publishing high-quality manuscripts focusing on the biology and applications of stem cell sions to Stem Cell Research, may cover all aspects of stem cells, including embryonic stem cells, tissue-specific stem cells, cancer stem cells, developmental studies, genomics and translational research.

Special focus of SCR is on mechanisms of. I ntroduction. Mouse embryonic stem (ES) cells are derived from the epiblast of preimplantation embryos [1, 2].ES cells are unique in their capacity to form all cells of the developing embryo and are useful in studies ranging from in vitro differentiation to knockout mice [2, 7].Therefore, ES cells are an important tool for both basic and applied research, and the efficient manipulation of.

p7: novel strategy for cell type specific gene expression through retroviral transduction of bone marrow stem cells Jie Yu Chung 1, Zeyad Nasa 1, Amit Joglekar 1 and Frank Alderuccio 1, 1 Autoimmunity Lab, Dept.

of Immunology, Central Clinical School, Monash University, AMREP, Commercial Road, Prahran. Nature today retracted two controversial papers on stem cells that it published in January 1, retractions — agreed to by all of the co-authors — come at the end of a whirlwind five.Stem cells express both unique and specific combinations of transcription factors, cell surface proteins, and cytoplasmic proteins.

Techniques used for stem cell analysis and characterization include flow cytometry, array-based analysis of the transcriptome, immunocytochemistry, western blots, and biomarker analysis.In this Commission, we argue that a combination of poor quality science, unclear funding models, unrealistic hopes, and unscrupulous private clinics threatens regenerative medicine's social licence to operate.

If regenerative medicine is to shift from mostly small-scale bespoke experimental interventions into routine clinical practice, substantial rethinking of the social contract that.