Telomerase-mediated life-span extension of human primary fibroblasts by human artificial chromosome (HAC) vector
Section snippets
Materials and methods
Cells and culture conditions. The 21ΔqHAC vector was stably maintained in CHO-K1 cells cultured as previously described [12]. The HFL1 is a primary cultured fibroblast derived from human fetal lung (RCB0521, passage 1, RIKEN, Tsukuba, Japan). The HFL1 cells were maintained in DMEM containing 20% FBS, and were cultured in collagen-I-coated tissue culture flasks/plates (Falcon) in this study.
Fluorescence in situ hybridization. Preparation of metaphase chromosomes and hybridization were carried
Construction of the hTERT-HAC and Flpe-induced hTERT expression in CHO cells
To construct HACs carrying hTERT expression cassettes, we employed the hChr.21-derived, mini-chromosomal vector (21ΔqHAC) devoid of most part of the q-arm [2]. The 21ΔqHAC was stably maintained in the chinese hamster ovary (CHO) cell clone (KH21). The silent hTERT expression cassette, hTERT(OFF), is composed of the following DNA elements: a human ubiquitin C (UbC) promoter [26]; a FRT-flanked transcribe-translate termination sequence (STOP sequence) [27], [28]; a hTERT/internal ribosomal entry
Acknowledgments
We thank Drs. Makoto Kakitani and Ayano Inoue for valuable discussions.
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2020, Experimental Cell ResearchCitation Excerpt :Moreover, life-span extension using hTERT reportedly did not yield unexpected property changes or malignant transformation [40]. As with top-down HAC [41], we introduced bottom-up HACs carrying the hTERT gene into normal human fibroblasts, and found that these fibroblasts acquired telomerase activity to stabilize telomere length, and extended life-span. Selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown clinical responses in patients with advanced non-small-cell lung cancer (NSCLC), especially in patients with tumors harboring activating EGFR mutations [42].
Human artificial chromosomes for pluripotent stem cell-based tissue replacement therapy
2020, Experimental Cell ResearchCitation Excerpt :Other studies confirmed that 21ΔpqHAC vector provides long-term therapeutic erythropoietin expression in primary human fibroblasts [13]. It was also shown with the use of the 21ΔqHAC harboring human telomerase reverse transcriptase (hTERT) gene that the expression of the gene allows the extension of the life span of human primary fibroblast [131]. The 21ΔqHAC vector containing osteopontin promoter-GFP reporter was successfully transferred into human MSCs by MMCT method.
Telomeres and telomerase in T cells of tumor immunity
2014, Cellular ImmunologyCitation Excerpt :Replicative senescence in various human cell types, including fibroblasts and endothelial cells, can be prevented by the ectopic expression of hTERT. Its gene, transformed into various human cells, results in the extension of their replicative life span without inducing changes associated with transformation [69,70]. Human T lymphocytes display a limited life span- about 30–50 population doublings (PD) when cultured in vitro [27], telomeres shortened with each round of cell division with age [6].
Strategies for immortalization of primary hepatocytes
2014, Journal of HepatologyHuman Telomerase Reverse Transcriptase Transfection Reduces Apoptosis in Human Penile Smooth Muscle Cells and Slows Down Cellular Aging
2012, Journal of Sexual MedicineCitation Excerpt :Nevertheless, telomerase has been found in cycling cells, although it seems to be present only at early stages of subculturing [23,24], and the replicative senescence of these cells can be bypassed by ectopic expression of hTERT with considerable extension of the culture life span [25]. Research has shown that the introduction or activation of the hTERT gene can activate telomerase in somatic cells, thereby maintaining telomere length or delaying telomere loss [26-28]. Bodnar et al. used vectors encoding human telomerase catalytic subunit to transfect two types of normal human somatic cells without telomerase activity, retinal pigment epithelial cells and foreskin fibroblasts.
A new chromosome 14-based human artificial chromosome (HAC) vector system for efficient transgene expression in human primary cells
2011, Biochemical and Biophysical Research CommunicationsCitation Excerpt :Accordingly, the host genome is not disrupted, and transgene expression can be sustained for a prolonged period without being subjected to the effects of the host genome. HACs have no size limit on the DNA sequence that can be inserted, thus, genome regulatory regions and multiple transgenes can be introduced into HACs to control the physiological expression of transgenes and cellular functions [1–4]. These features of HACs are desirable characteristics for gene delivery vectors which can allow them to overcome various issues associated with viral and non-viral vector systems.