Immortalization of Rat Embryo Fibroblasts by a 3′-Untranslated Region (original) (raw)
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Establishment of murine cell lines by constitutive and conditional immortalization
Journal of Biotechnology, 2005
Mouse cell lines were immortalized by introduction of specific immortalizing genes. Embryonic and adult animals and an embryonal stem cell line were used as a source of primary cells. The immortalizing genes were either introduced by DNA transfection or by ecotropic retrovirus transduction. Fibroblasts were obtained by expression of SV40 virus large T antigen (TAg). The properties of the resulting fibroblast cell lines were reproducible, independent of the donor mouse strains employed and the cells showed no transformed properties in vitro and did not form tumors in vivo. Endothelial cell lines were generated by Polyoma virus middle T antigen expression in primary embryonal cells. These cell lines consistently expressed relevant endothelial cell surface markers. Since the expression of the immortalizing genes was expected to strongly influence the cellular characteristics fibroblastoid cells were reversibly immortalized by using a vector that allows conditional expression of the TAg. Under inducing conditions, these cells exhibited properties that were highly similar to the properties of constitutively immortalized cells. In the absence of TAg expression, cell proliferation stops. Cell growth is resumed when TAg expression is restored. Gene expression profiling indicates that TAg influences the expression levels of more than 1000 genes that are involved in diverse cellular processes. The data show that conditionally immortalized cell lines have several advantageous properties over constitutively immortalized cells.
Toxicology letters, 1993
Recent advances using somatic cell genetic approaches have provided a convincing body of evidence that the senescence of mammalian cells in culture is controlled by a small group of genes, one or more of which are functionally deleted in the process of immortalization. Microcell-mediated mono-chromosomal transfer methods should permit precise mapping of these genes to specific chromosomal regions. Cloning of senescence genes, using either conventional 'positional cloning' techniques or retroviral insertion mutagenesis, is now a realistic possibility. The leap in our understanding of the molecular genetic events driving the alternative cellular states of limited proliferative capacity and immortality, which such advances should precipitate, will finally permit the question of the role of cell immortalization in cancer to be addressed, and may open the door to the design of new modes of cancer therapy. In addition, the precise mechanism underlying the wide difference in transf...
Genetic changes during immortalization of human cells
Radiation Oncology Investigations, 1995
Spontaneous immortalization of fibrobIasts containing an inherited mutation of one p53 allele was associated with loss of the wild-type p53 allele and loss of ~1 6~~ gene expression, but this combination of genetic events was insufficient for immortalization. Loss of ~1 6~~ expression appears to be an alternative to loss of functional retinoblastoma gene product. In all cell lines studied, immortalization was associated either with stabilization of telomere length in the presence of telomerase activity or with the acquisition of long and heterogeneous telomeres in the absence of detectable telomerase. The role of other genes, such as human stanniocalcin, i n the immortalization process is under investigation.
AGE, 1999
Model systems implementing various approaches to immortalize cells have led toward further understanding of replicative senescence and carcinogenesis. Human diploid cells have a limited life span, termed replicative senescence. Because cells are terminally growth arrested during replicative senescence, it has been suggested that it acts as a tumor suppression mechanism as tumor cells exhibit an indefinite life span and are immortal. The generation of immortal cells lines, by the introduction of SV40 and human papillomavirus (HPV) sequences into cells, has provided invaluable tools to dissect the mechanisms of immortalization. We have developed matched sets of nonimmortal and immortal SV40 cell lines which have been useful in the identification of novel growth suppressor genes (SI=N6) as well as providing a model system for the study of processes such as cellular aging, apoptosis, and telomere stabilization. Thus, their continued use is anticipated to lead to insights into other processes, which are effected by the altered expression of oncogenes and growth suppressors.
Biology of the Cell, 1990
The cellular immortalization activity of cloned genes can be identified either in a colony-forming assa of transfected primary rat embryo fibroblasts or in a co-operation assay together with ras. However the demonstration of immortalization activities carried by cellular genes has not been reported. Here we establish that SV40 early genes integrated in genomic DNAs can be stably transferred into rat embryo fibroblasts and selected via their immortalization activity. Attempts to extend this assay to the identification of dominant genes putatively involved in the immortality of several other immortal post-crisis or tumor cells have been unsuccessful suggesting that the immortal phenotype can be brought about through different pathways.
Cloning and Identification of Genes That Associate with Mammalian Replicative Senescence
Experimental Cell Research, 1998
lated phenomena whose understanding at the molecu-Cellular senescence and limited proliferative capac-lar level is still incomplete. Whereas a normal diploid ity of normal diploid cells has a dominant phenotype cell has a limited capacity to divide in vivo and in vitro, over immortality of cancerous cells, suggesting its rega cell that has been genetically transformed either ulation by the expression of a set of genes. In order to spontaneously or by various physical, chemical, or bioisolate the genes that associate with senescence, we logical agents has escaped the regulatory mechanisms have employed a clonal system of conditional SV40 T of limited growth and has a tendency to divide indefiantigen rat embryo fibroblast cell lines which undergo nitely and to form a clone of cancerous cells. While senescence upon T antigen inactivation. Construction cellular mortality is characterized by a progressive cesof cDNA libraries from two conditional cell lines and sation of cell growth manifested in cell culture by senesapplication of differential screening and subtractive cence, cellular immortalization is the escape from sehybridization techniques have resulted in the cloning nescence as a result of multiple mechanisms involving of eight senescence-induced genes (SGP-2/Apo J, genetic and epigenetic changes [reviewed in 1, 2].