Establishment and characterization of conditionally immortalized cells from the mouse urogenital ridge

1996 ◽  
Vol 109 (5) ◽  
pp. 899-909 ◽  
Author(s):  
B. Capel ◽  
J.R. Hawkins ◽  
E. Hirst ◽  
D. Kioussis ◽  
R. Lovell-Badge
Keyword(s):  
Primordial Germ Cells ◽  
Cell Types ◽  
T Antigen ◽  
Temperature Sensitive ◽  
Sv40 Large T Antigen ◽  
In Vitro Morphogenesis ◽  
Immortalized Cells ◽  
Clonal Lines

Cell cultures from the urogenital ridge have been established to facilitate the study of the regulation and downstream interactions of Sry in mammalian sex determination. Cells have been explanted from transgenic mice carrying a temperature sensitive SV40 large T-antigen, and established in ongoing cultures. Analysis of the cells in these cultures at the electron microscope level reveals multiple cell types that compare to the cell types found in vivo during this period of development. Primordial germ cells, that are simultaneously explanted in the course of these experiments, also survive in culture. The explants undergo a morphogenetic organization into branching cord-like structures when cells are trypsinized and plated in extracellular matrix (Matrigel). We analyzed the expression of a number of molecular markers of the fetal gonad during monolayer culture, during in vitro morphogenesis in Matrigel, and in clonal lines derived from the complex explants. This analysis included Sry which is found to be expressed in some cultures from XY urogenital ridges that have been maintained for as long as 8 months.

In vitro and in vivo characterisation of glial cells immortalised with a temperature sensitive SV40 T antigen-containing retrovirus

1994 ◽  
Vol 37 (2) ◽  
pp. 182-196 ◽  
Author(s):  
M. Jung ◽  
A. J. Crang ◽  
W. F. Blackemore ◽  
D. Hoppe ◽  
H. Kettenmann ◽  
...  
Keyword(s):  
Glial Cells ◽  
T Antigen ◽  
Temperature Sensitive ◽  
Sv40 T Antigen

scholarly journals Generation of Sheep Induced Pluripotent Stem Cells With Defined DOX-Inducible Transcription Factors via piggyBac Transposition

2021 ◽  
Vol 9 ◽  
Author(s):  
Moning Liu ◽  
Lixia Zhao ◽  
Zixin Wang ◽  
Hong Su ◽  
Tong Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
T Antigen ◽  
Sv40 Large T Antigen ◽  
Adult Individual ◽  
Induced Pluripotent

Pluripotent stem cells (PSCs) have the potential to differentiate to all cell types of an adult individual and are useful for studying mammalian development. Establishing induced pluripotent stem cells (iPSCs) capable of expressing pluripotent genes and differentiating to three germ layers will not only help to explain the mechanisms underlying somatic reprogramming but also lay the foundation for the establishment of sheep embryonic stem cells (ESCs) in vitro. In this study, sheep somatic cells were reprogrammed in vitro into sheep iPSCs with stable morphology, pluripotent marker expression, and differentiation ability, delivered by piggyBac transposon system with eight doxycycline (DOX)-inducible exogenous reprogramming factors: bovine OCT4, SOX2, KLF4, cMYC, porcine NANOG, human LIN28, SV40 large T antigen, and human TERT. Sheep iPSCs exhibited a chimeric contribution to the early blastocysts of sheep and mice and E6.5 mouse embryos in vitro. A transcriptome analysis revealed the pluripotent characteristics of somatic reprogramming and insights into sheep iPSCs. This study provides an ideal experimental material for further study of the construction of totipotent ESCs in sheep.

Simian virus 40 small t antigen cooperates with mitogen-activated kinases to stimulate AP-1 activity

1994 ◽  
Vol 14 (9) ◽  
pp. 6244-6252
Author(s):  
J A Frost ◽  
A S Alberts ◽  
E Sontag ◽  
K Guan ◽  
M C Mumby ◽  
...  
Keyword(s):  
Simian Virus 40 ◽  
Cell Types ◽  
Simian Virus ◽  
T Antigen ◽  
Mitogen Activated Protein Kinase ◽  
Early Gene ◽  
Mitogen Activated Protein ◽  
Small T Antigen

The simian virus 40 small tumor antigen (small t) specifically interacts with protein phosphatase type 2A (PP2A) in vivo and alters its catalytic activity in vitro. Among the substrates for PP2A in vitro are the activated forms of MEK and ERK kinases. Dephosphorylation of the activating phosphorylation sites on MEK and ERKs by PP2A in vitro results in a decrease in their respective kinase activities. Recently, it has been shown that overexpression of small t in CV-1 cells results in an inhibition of PP2A activity toward MEK and ERK2 and a constitutive upregulation of MEK and ERK2 activity. Previously, we have observed that overexpression of either ERK1, MEK1, or a constitutively active truncated form of c-Raf-1 (BXB) is insufficient to activate AP-1 in REF52 fibroblasts. We therefore examined whether overexpression of small t either alone or in conjunction with ERK1, MEK1, or BXB could activate AP-1. We found that coexpression of small t and either ERK1, MEK1, or BXB resulted in an increase in AP-1 activity, whereas expression of either small t or any of the kinases alone did not have any effect. Similarly, coexpression of small t and ERK1 activated serum response element-regulated promoters. Coexpression of kinase-deficient mutants of ERK1 and ERK2 inhibited the activation of AP-1 caused by expression of small t and either MEK1 or BXB. Coexpression of an interfering MEK, which inhibited AP-1 activation by small t and BXB, did not inhibit the activation of AP-1 caused by small t and ERK1. In contrast to REF52 cells, we observed that overexpression of either small or ERK1 alone in CV-1 cells was sufficient to stimulate AP-1 activity and that this stimulation was not enhanced by expression of small t and ERK1 together. These results show that the effects of small t on immediate-early gene expression depend on the cell type examined and suggest that the mitogen-activated protein kinase activation pathway is distinctly regulated in different cell types.

Molecular pathophysiology of glucagon-SV40 T antigen transgenic mice

1994 ◽  
Vol 267 (5) ◽  
pp. E629-E635 ◽  
Author(s):  
D. J. Drucker
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Dna Sequences ◽  
Large Bowel ◽  
Biological Activities ◽  
T Antigen ◽  
Sv40 Large T Antigen ◽  
Molecular Determinants

The gene encoding proglucagon is expressed in the pancreas, intestine, and brain. The molecular determinants of proglucagon gene expression and the biological activities of the proglucagon-derived peptides (PGDPs) have been examined using transgenic mice harboring a glucagon-SV40 large T antigen (GLUTag) transgene. These experiments have delineated DNA sequences important for intestinal-specific proglucagon gene transcription. GLUTag mice develop neuroendocrine tumors of the pancreas and large bowel, leading to elevated plasma levels of the PGDPs and suppression of endogenous proglucagon gene expression. Transplantation of the large bowel tumors subcutaneously into nude mice provides additional evidence for inhibition of endogenous pancreatic proglucagon gene expression by one or more of the tumor-derived PGDPs. The large bowel GLUTag tumors exhibit abnormalities in the posttranslational processing of proglucagon. GLUTag tumors may be passaged in vivo and in vitro and have been used to generate stable cell lines that express the proglucagon gene at high levels. Taken together, these studies highlight the utility of transgenic systems for the physiological analysis of hormone action and the molecular determinants of peptide hormone gene expression.

Interferon-gamma regulation of Clara cell gene expression: in vivo and in vitro

1997 ◽  
Vol 272 (6) ◽  
pp. L1142-L1151 ◽  
Author(s):  
S. M. Magdaleno ◽  
G. Wang ◽  
K. J. Jackson ◽  
M. K. Ray ◽  
S. Welty ◽  
...  
Keyword(s):  
Interferon Gamma ◽  
Potential Interaction ◽  
T Antigen ◽  
Clara Cell ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Sv40 Large T Antigen ◽  
Ifn Gamma

This report demonstrates that Clara cell 10-kDa protein (CC10) mRNA levels are regulated by interferon-gamma (IFN-gamma). An analysis of total lung RNA from mice given IFN-gamma intratracheally showed increased levels of CC10 mRNA compared to control animals but no significant increases in surfactant proteins B and C. These results were confirmed in a Clara cell line, mtCC1-2, generated from the lungs of a transgenic mouse expressing the SV40 large T antigen under the control of a Clara cell-specific promoter. Significant increases in mtCC1-2 CC10 mRNA levels were observed in a time- and a dose-dependent manner. The expression of transacting factors hepatocyte nuclear factors 3 alpha and 3 beta (HNF-3 alpha and HNF-3 beta) were also analyzed, and a transient increase in the expression of HNF-3 beta but not HNF-3 alpha was detected. Deoxyribonuclease I footprint analysis identified a signal transducer and activator of transcription (STAT) binding site (at nucleotides -293 to -284 of CC10) adjacent to two thyroid transcription factor-1 (TTF-1) binding sites, suggesting a potential interaction between STAT1 and TTF-1. This report reinforces the hypothesis that CC10 functions as an anti-inflammatory protein and that increases in CC10 protein may provide additional protection from inflammation and disease in the lung.

scholarly journals TMOD-16. A NOVEL ADENOVIRAL-PERMISSIVE, IMMUNOCOMPETENT HAMSTER GLIOMA MODEL TO EVALUATE ONCOLYTIC ADENOVIRAL THERAPY

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii231-ii231
Author(s):  
Lynette Phillips ◽  
Joy Gumin ◽  
Shoudong Li ◽  
Marc Daou ◽  
Daniel Ledbetter ◽  
...  
Keyword(s):  
Immune Response ◽  
Viral Replication ◽  
Tumor Infiltrating Lymphocytes ◽  
Peripheral Blood Lymphocytes ◽  
T Antigen ◽  
Sv40 Large T Antigen ◽  
Oncolytic Adenoviruses ◽  
Glioma Model

Abstract Oncolytic adenoviruses, including Delta-24-RGD, target tumors by direct tumor cell oncolysis and by activation of an anti-tumor immune response. Due to the species selectivity of oncolytic adenoviruses, there is currently no single preclinical animal model of glioma that supports viral replication, tumor oncolysis, and virus-mediated immune responses. To address this gap, we took advantage of the Syrian hamster to develop the first glioma model that is both adenovirus replication-permissive and immunocompetent. Hamster glioma stem-like cells (GSCs), transformed by forced expression of hTERT, SV40 large T antigen, and h-RasV12, reproducibly form intracranial tumors in hamsters. In vitro, electron microscopy and cytopathic effect assays demonstrated that hamster GSCs supported viral replication and were susceptible to Delta-24-RGD-mediated cell death. In vivo, hamster GSCs consistently developed into highly proliferative tumors resembling high-grade gliomas. Following intratumoral delivery of Delta-24-RGD, immunohistochemistry for viral proteins demonstrated viral infectivity and replication in hamster gliomas. Flow cytometry revealed increased T cell infiltration in Delta-24-RGD-infected tumors. Delta-24-RGD treatment of tumor-bearing hamsters led to significantly increased survival compared with hamsters treated with PBS. Using this model, we evaluated the effects of corticosteroid-mediated immunosuppression on Delta-24-RGD efficacy. Dexamethasone treatment significantly decreased peripheral blood lymphocytes, decreased tumor-infiltrating lymphocytes, and suppressed the levels of serum anti-adenovirus antibodies. Dexamethasone reduced the number of long-term survivors and decreased the median survival (50 days for Delta-24-RGD + dexamethasone vs undetermined for Delta-24-RGD alone). In summary, we have developed the first adenovirus-permissive, immunocompetent hamster glioma model, addressing a critical need for a model in which to study the role of direct oncolysis in driving immune mediated viral clearance versus driving an antiglioma immune response. Understanding these mechanisms is critical to optimizing the success of oncolytic adenoviral therapy in the clinic.

scholarly journals Invasive behaviour of mouse primordial germ cells in vitro

1986 ◽  
Vol 86 (1) ◽  
pp. 133-144
Author(s):  
D. Stott ◽  
C.C. Wylie
Keyword(s):  
Germ Cells ◽  
Primordial Germ Cells ◽  
Time Lapse ◽  
Cell Types ◽  
Fluorescent Labelling ◽  
Mouse Embryo Fibroblast ◽  
Substrate Interaction ◽  
Close Contacts

We have isolated migrating primordial germ cells (PGCs) from 10.5-day mouse embryos and studied their behaviour when cultured on a mouse embryo fibroblast (STO) cell line. Living and fixed PGCs were identified by fluorescent labelling with a monoclonal antibody specific for PGCs in the culture system used. The behaviour of the cells was studied using interference reflexion microscopy (IRM) and time-lapse video cinematography. The IRM pattern displayed by PGCs is typical of highly motile cell types, the cells lack focal contacts and possess large areas of close contacts indicative of weak membrane to substrate interaction. The PGCs exhibit relatively high rates of translocation and lack contact inhibition. They were observed to underlap STO cells in subconfluent monolayers and to penetrate between the cells of confluent monolayers, becoming located between the monolayer and its substrate. These observations support the hypothesis that migrating mouse PGCs are inherently motile and are able transiently to disrupt the adhesion of surrounding cells. These results suggest that PGCs actively migrate to the developing gonad in vivo.

scholarly journals Homologous and nonhomologous recombination in monkey cells.

1983 ◽  
Vol 3 (6) ◽  
pp. 1040-1052 ◽  
Author(s):  
S Subramani ◽  
P Berg
Keyword(s):  
Mammalian Cells ◽  
Plaque Assay ◽  
Simian Virus 40 ◽  
Helper Virus ◽  
Simian Virus ◽  
T Antigen ◽  
Temperature Sensitive ◽  
Sv40 Large T Antigen ◽  
Nonhomologous Recombination

Though recombinational events are important for the proper functioning of most cells, little is known about the frequency and mechanisms of recombination in mammalian cells. We have used simian virus 40 (SV40)-pBR322 hybrid plasmids constructed in vitro as substrates to detect and quantitate intramolecular homologous and nonhomologous recombination events in cultured monkey cells. Excision of wild-type or defective SV40 DNAs by recombination from these plasmids was scored by the viral plaque assay, in either the absence or the presence of DNA from a temperature-sensitive helper virus. Several independent products of homologous and nonhomologous recombination have been isolated and characterized at the DNA sequence level. We find that neither DNA replication of the recombination substrate nor SV40 large T antigen is essential for either homologous or nonhomologous recombination involving viral or pBR322 sequences.

The biology of JC polyomavirus

2017 ◽  
Vol 398 (8) ◽  
pp. 839-855 ◽  
Author(s):  
Benedetta Assetta ◽  
Walter J. Atwood
Keyword(s):  
Demyelinating Disease ◽  
Cell Types ◽  
International Committee ◽  
T Antigen ◽  
Oncogenic Viruses ◽  
Jc Polyomavirus ◽  
Tumor Virus ◽  
Immunomodulatory Therapies

Abstract JC polyomavirus (JCPyV) is the causative agent of a fatal central nervous system demyelinating disease known as progressive multifocal leukoencephalopathy (PML). PML occurs in people with underlying immunodeficiency or in individuals being treated with potent immunomodulatory therapies. JCPyV is a DNA tumor virus with a double-stranded DNA genome and encodes a well-studied oncogene, large T antigen. Its host range is highly restricted to humans and only a few cell types support lytic infection in vivo or in vitro. Its oncogenic potential in humans has not been firmly established and the international committee on oncogenic viruses lists JCPyV as possibly carcinogenic. Significant progress has been made in understanding the biology of JCPyV and here we present an overview of the field and discuss some important questions that remain unanswered.

Sign in / Sign up

Export Citation Format

Share Document