Sleeping Beauty Transposon-based System for Rapid Generation of HBV-replicating Stable Cell Lines

MCPIP2 is the least known member of the MCPIP family of proteins. Recently we have found that it is a new RNase involved in transcript turnover. However, the full spectrum of its cellular targets is still unidentified. To discover transcripts which are regulated by this protein we have employed Sleeping Beauty transposons. This tool allows for rapid generation of a stable transgenic cell line with inducible expression of the desired gene. In this study, we analysed how the Sleeping Beauty system itself influences expression of chosen genes, namely IL-6, Regnase-1 and VEGF. We found that the system alone may influence expression of IL-6. Our results indicate that Sleeping Beauty transposons should be used with caution in studies that are focused on changes in the transcript level.

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Rapid Generation of Stable Cell Lines Expressing Corticotropin-Releasing Hormone Receptor for Drug Discovery

Protein Expression and Purification ◽

10.1006/prep.1996.0701 ◽

1997 ◽

Vol 9 (3) ◽

pp. 301-308 ◽

Cited By ~ 12

Author(s):

Robert A. Horlick ◽

Karen Sperle ◽

Leah A. Breth ◽

Christian C. Reid ◽

Emily S. Shen ◽

...

Keyword(s):

Drug Discovery ◽

Cell Lines ◽

Hormone Receptor ◽

Corticotropin Releasing Hormone ◽

Releasing Hormone ◽

Stable Cell Lines ◽

Rapid Generation

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Rapid Generation of Stable Cell Lines Expressing High Levels of Erythropoietin, Factor VIII, and an Antihuman CD20 Antibody Using Lentiviral Vectors

Human Gene Therapy Methods ◽

10.1089/hgtb.2013.002 ◽

2013 ◽

Vol 24 (4) ◽

pp. 214-227 ◽

Cited By ~ 9

Author(s):

Lajos Baranyi ◽

Christopher B. Doering ◽

Gabriella Denning ◽

Richard E. Gautney ◽

Kyle T. Harris ◽

...

Keyword(s):

Factor Viii ◽

Cell Lines ◽

Lentiviral Vectors ◽

Stable Cell Lines ◽

Cd20 Antibody ◽

Rapid Generation

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292 GENERATION OF MOUSE INDUCED PLURIPOTENT STEM CELLS FROM VARIOUS GENETIC BACKGROUND BY SLEEPING BEAUTY TRANSPOSON-MEDIATED GENE TRANSFER

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab292 ◽

2011 ◽

Vol 23 (1) ◽

pp. 243 ◽

Cited By ~ 1

Author(s):

S. Muenthaisong ◽

O. Ujhelly ◽

E. Varga ◽

A. C. Carstea ◽

Z. Ivics ◽

...

Keyword(s):

Cell Lines ◽

Pluripotent Stem Cells ◽

Ips Cells ◽

Sleeping Beauty ◽

Embryoid Bodies ◽

Es Cell ◽

Differentiation Potential ◽

Ips Cell ◽

Sleeping Beauty Transposon ◽

Induced Pluripotent

Induced pluripotent stem (iPS) cell technology allows the reprogramming of somatic cells to a pluripotent state; however, it requires viral gene transduction and permanent existence of the exogenous genes in the genome, which is a potential risk for abnormalities in the derived iPS cells. Recently, there was report that iPS cells have been made with piggyBack transposon. Here, we first reported that nonviral transfection of a Sleeping Beauty transposon, which comprises c-Myc, Klf-4, Oct3/4 (Pou5f1), and Sox-2, can reprogram mouse fibroblasts from 3 different genetic backgrounds: ICR (outbred), C57BL/6 (inbred), and F1 hybrid (C57BL/6 × DBA/2J), with parallel robust expression of all exogenous (c-Myc, Klf-4, Oct3/4, and Sox-2) and endogenous (e.g. Nanog) pluripotency genes. The iPS cells were cultured under standard conditions with promotion of differentiate by withdrawal of leukemia inhibitory factor. We chose 6 cloned of each line that exhibited characteristics typical for undifferentiated embryonic stem (ES) cell: ES-cell-like morphology, alkaline phosphatase positivity, and gene expression pattern [quantitative real-time PCR and immunofluorescence of ES cell markers (e.g. Oct-4, SSEA1, Nanog]. Furthermore, cells were able to form embryoid bodies and beat rhythmically and expressed cardiac markers assayed by immunofluorescence (e.g. cardiac Troponin T, desmin). In vivo testing of iPS cell lines for their developmental potential (diploid and tetraploid embryo complementation assay) is currently underway. The iPS cell lines generated from the ICR strain appeared the earliest in time (ICR-d11, F1 day-2 and Bl6-d12), with higher efficiency than colonies from the other 2 backgrounds. The differentiation potential of the iPS lines derived from the 3 genetic backgrounds was similar. Interestingly, the ICR-iPS lines had higher differentiation potential than did the ICR-ES cell lines: the rate of embryoid bodies forming rhythmically beating cardiomyocytes was 4% in ICR-ES and 79% in ICR-iPS cells, respectively. Our results suggest that the iPS technology provide a new tool to generate pluripotent stem cells from genetic backgrounds where good-quality ES cell generation is difficult. These studies provide new insights into virus-free iPS technology and contribute to defining future cell-based therapies, drug screening methods, and production of transgenic animals with genetically modified iPS cells. This study was financed by EU FP6 (CLONET, MRTN-CT-2006-035468), EU FP7 (PartnErS, PIAP-GA-2008-218205; InduHeart, PEOPLE-IRG-2008-234390; InduVir, PEOPLE-IRG-2009-245808; InduStem, PIAP-GA-2008-230675; PluriSys, HEALTH-2007-B-223485); NKTH-OTKA-EU FP7-HUMAN-2009-MB08-C 80205, and NKTH/KPI (Jedlik NKFP_07_1-ES2HEART-HU OM-00202-2007).

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CD200 Expression on Multiple Myeloma Cells Induces Attenuation of T Cell-Mediated Cytotoxicity Via DOK2

Blood ◽

10.1182/blood-2021-153741 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1587-1587

Author(s):

Pooja Shah ◽

Thorsten Stuehmer ◽

Daniela Bruennert ◽

Umair Munawar ◽

Ellen Leich ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Cell Lines ◽

Sleeping Beauty ◽

Luciferase Assay ◽

Vector System ◽

Activated T Cells ◽

Blocking Antibody ◽

Sleeping Beauty Transposon

Abstract Introduction The CD200/CD200 receptor (CD200R) axis is known to exert immunoregulatory effects in myeloid-derived cells and constitutes a putative immune checkpoint in hematological malignancies, in which CD200 expression is associated with poor prognosis. In multiple myeloma (MM), CD200 is expressed in the majority of patient-derived primary cells. However, its functional importance as well as the related downstream mechanism upon CD200 ligand binding to its CD200R on T cells are not well understood. In this study, we analyze the functional role of CD200 as a potential immune checkpoint in MM and decipher the mechanism of CD200-mediated immune escape. Methods Primary MM cells and MM cell lines were analyzed for CD200 surface expression by flow cytometry. To overexpress CD200 on non-expressing MM cell lines we used a Sleeping Beauty transposon vector system. CD200+/- MM cell lines L363, U266 and MM.1s were co-cultured with CD3/CD28-activated healthy donor T cells. T cell-mediated cytotoxicity in these co-cultures was assessed with flow cytometry and/or luciferase assay. Moreover, to analyze the effects of CD200R activation on downstream signaling pathways, activated T cells were treated with recombinant human CD200 (rhCD200) and/or anti-CD200 blocking antibody and subsequently, Western blotting was performed. Results Of n=120 primary MM samples (n=120) analyzed, CD200 protein expression could be detected in ca. 75 % of the cases. In contrast, all n=9 MM cell lines tested neither displayed surface nor cytoplasmic CD200 expression. Therefore, using a Sleeping Beauty transposon vector system we stably expressed CD200 on MM cell lines for further analyses. In the presence of CD200-expressing MM cells up to 50% decrease in CD3+ T cell-mediated cytotoxicity against MM cells was observed in flow cytometry and luciferase assay. Proliferation rates of MM cell lines remained unchanged regardless of the level of CD200 overexpression as determined by Alamar blue assays. In myeloid-derived cells, CD200R directly interacts with docking protein-2 (DOK2). In activated T cells, we observed DOK2 phosphorylation upon CD200 binding when treated with rhCD200 in a time- and concentration-dependent manner. Applying an anti-CD200 blocking antibody, this effect could be reversed, thus revealing a possible mechanism for the observed attenuation of T cell function. Conclusion Our study shows that anti-MM cytotoxicity from primary healthy donor CD3+ T cells is attenuated by CD200 expression on MM cells. We also demonstrate that this inhibitory mechanism in CD3+ T cells is mediated via DOK2, providing a potential target for immunotherapeutic approaches in MM. Disclosures Einsele: Janssen, Celgene/BMS, Amgen, GSK, Sanofi: Consultancy, Honoraria, Research Funding.

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Faculty Opinions recommendation of Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1026892.329485 ◽

2005 ◽

Author(s):

Donald Rio

Keyword(s):

Sleeping Beauty ◽

Sleeping Beauty Transposon

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Cell-Based Gene Therapy Using the Sleeping Beauty Transposon System Reduces Neovascularisation in Rats

SSRN Electronic Journal ◽

10.2139/ssrn.3408024 ◽

2019 ◽

Author(s):

Maria Hernandez ◽

Sergio Recalde ◽

Laura Garcia-Garcia ◽

Jaione Bezunartea ◽

Csaba Miskey ◽

...

Keyword(s):

Gene Therapy ◽

Sleeping Beauty ◽

Sleeping Beauty Transposon

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Rapid Generation of Single-Tumor Spheroids for High-Throughput Cell Function and Toxicity Analysis

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057106292763 ◽

2006 ◽

Vol 11 (8) ◽

pp. 922-932 ◽

Cited By ~ 321

Author(s):

Andrea Ivascu ◽

Manfred Kubbies

Keyword(s):

Tumor Cell ◽

Suspension Culture ◽

Cell Lines ◽

Cell Function ◽

Tumor Cell Lines ◽

Proliferating Cells ◽

Basement Membrane Extract ◽

Rapid Generation ◽

Size Heterogeneity

Spheroids are widely used in biology because they provide an in vitro 3-dimensional (3D) model to study proliferation, cell death, differentiation, and metabolism of cells in tumors and the response of tumors to radiotherapy and chemotherapy. The methods of generating spheroids are limited by size heterogeneity, long cultivation time, or mechanical accessibility for higher throughput fashion. The authors present a rapid method to generate single spheroids in suspension culture in individual wells. A defined number of cells ranging from 1000 to 20,000 were seeded into wells of poly-HEMA-coated, 96-well, round-or conical-bottom plates in standard medium and centrifuged for 10 min at 1000 g. This procedure generates single spheroids in each well within a 24-h culture time with homogeneous sizes, morphologies, and stratification of proliferating cells in the rim and dying cells in the core region. Because a large number of tumor cell lines form only loose aggregates when cultured in 3D, the authors also performed a screen for medium additives to achieve a switch from aggregate to spheroid morphology. Small quantities of the basement membrane extract Matrigel, added to the culture medium prior to centrifugation, most effectively induced compact spheroid formation. The compact spheroid morphology is evident as early as 24 h after centrifugation in a true suspension culture. Twenty tumor cell lines of different lineages have been used to successfully generate compact, single spheroids with homogenous size in 96-well plates and are easily accessible for subsequent functional analysis.

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