The telomere lengths also were compared for fetal mesenchymal, newborn mesenchymal, and adult fibroblast cells during subcultures. Telomere lengths of pig cells, regardless of the age of the animals, were longer than those of human fibroblasts (Figure (Figure1A, 1A, B). Adult pig fibroblasts had telomere lengths similar to those of newborn fibroblasts. Telomere lengths of newborn fibroblasts were slightly shorter than those of fetal fibroblasts during their early passages. ![]() Telomere lengths shown as TRFs decrease during subculture of pig primary cellsįibroblasts and mesenchymal cells derived from the bone marrow of fetal (embryonic day 50 abbreviated as FF and FM, respectively) and newborn (7 or 8 days old NF and NM) pigs, as well as fibroblasts from adult pigs (3–4 months of age AF), during their early passages, did not show significantly different telomere lengths (Figure (Figure1A, 1A, B) by Southern blot analysis. We sought to measure pig telomeres by comparing three methods, Southern blot, Q-FISH, and qPCR, and to characterize pig telomeres in relevance to cellular senescence during subculture of pig primary cells. Moreover, the precise characteristics of pig telomeres and their roles in cellular senescence and immortalization remain elusive. Thus far, quantitative measurement of telomeres at the level of individual chromosomes has not been performed in porcine cells. The shortest telomeres, or fragile telomeres, may reflect DNA-damage response signals in senescent human cells. Telomeres were recently found to resemble fragile sites. Consistently, chromosome arms carrying the shortest telomeres are the first to be unstable. However, it is not the average telomere length but rather the shortest telomere that constitutes telomere dysfunction and that becomes a major determinant of the onset of senescence. TRFs show distribution of telomeres in smear gels by Southern blots, and only average telomere length is estimated by this approach. TRF measurement by Southern blot was employed to examine telomere lengths in cloned pigs. ![]() Pig telomeres have been revealed by fluorescence in situ hybridization using human telomere repeat probe (TTAGGG)n and primed in situ DNA synthesis (PRINS), but telomere measurement by either method was not quantitative. The major quantitative methods available for telomere measurement include length distribution of TRFs by Southern blot, quantitative fluorescence in situ hybridization (Q-FISH) that shows individual telomere lengths of metaphase spreads, mean telomere length by quantitative PCR (qPCR), and PCR of single telomere lengths (STELA). The terminal restriction fragments (TRFs) from pig cells (9–50 kb) are longer than those of human cells (10–20 kb) but shorter than those of laboratory mice (30–200 kb). Pig telomeres share the conserved TTAGGG sequence of mouse and human telomeres. Also, primary porcine cells have been genetically engineered to induce tumors in size similar to those observed clinically, and may provide a robust cancer model for preclinical studies. Pigs have been considered as an ideal organ provider for xeno-transplantation and also as appropriate large animal models for preclinical tests and study of human diseases, including cardiovascular disease, diabetes, infectious disease, and cancer, and stem cell therapy, owing to the similarities in anatomy and physiology between pigs and humans. Other mammals, including dogs, primates, and sheep, have also been compared for telomere biology and function. It was proposed that the mouse may not be the best animal model for study of human telomere biology, as fundamental differences exist between human and mouse telomere biology. ![]() The observed senescence of mouse cells in culture may not be related to telomere shortening, but rather to changing culture condition, such as excessive oxidative stress. Mouse cells may have telomere damage signaling pathways different from those of humans. ![]() Normal human cells, including the stem cells of renewal tissues, show progressive telomere shortening with cell division until a subset of telomeres reach a critically short length, inducing DNA-damage response and replicative senescence or cell aging. Telomere sequence is conserved among mammals. Telomeres consist of (TTAGGG)n repeats and associated proteins at the end of chromosomes in mammalian cells and function in the maintenance of genomic stability to protect the chromosomes from degradation and end-to-end fusion.
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