International Journal of Genetics and Genomics
Volume 7, Issue 3, September 2019, Pages: 60-68
Received: Jul. 1, 2019;
Accepted: Aug. 3, 2019;
Published: Aug. 23, 2019
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Fang Wang, Department of Obstetrics and Gynecology, Laboratory of Reproductive Medicine and NYU Fertility Center, NYU Langone Medical Center, New York, USAS
Leroy Robinson, Department of Obstetrics and Gynecology, Laboratory of Reproductive Medicine and NYU Fertility Center, NYU Langone Medical Center, New York, USAS
Yael Kramer, Department of Obstetrics and Gynecology, Laboratory of Reproductive Medicine and NYU Fertility Center, NYU Langone Medical Center, New York, USAS
Keri Kalmbach, Department of Obstetrics and Gynecology, Laboratory of Reproductive Medicine and NYU Fertility Center, NYU Langone Medical Center, New York, USAS
Paula Andrea Navarro, Department of Obstetrics and Gynecology, Laboratory of Reproductive Medicine and NYU Fertility Center, NYU Langone Medical Center, New York, USAS; Department of Obstetrics and Gynecology, USP Ribeirao Preto, Ribeirao Preto, Sao Paulo, Brazil
Ricardo Pimentel, Department of Obstetrics and Gynecology, Laboratory of Reproductive Medicine and NYU Fertility Center, NYU Langone Medical Center, New York, USAS
Xinghua Victor Pan, Department of Biochemistry and Molecular Biology, Southern Medical University, Ghuangzhou, China; Department of Genetics, Yale University School of Medicine, New Haven, USA
Sherman Weissman, Department of Genetics, Yale University School of Medicine, New Haven, USA
Lin Liu, Department of Obstetrics and Gynecology, Laboratory of Reproductive Medicine and NYU Fertility Center, NYU Langone Medical Center, New York, USAS; College of Life Sciences, Nankai University, Tianjin, China
David Keefe, Department of Obstetrics and Gynecology, Laboratory of Reproductive Medicine and NYU Fertility Center, NYU Langone Medical Center, New York, USAS
Increasing evidence demonstrates that shortest more than mean telomere length predicts telomere dysfunction and genomic instability in association with a number of conditions, including cell senescence, aging and tumorigenesis. We developed Universal Single Cell Single Telomere Length Analysis (USC-STELA), based on a PCR-amplification and southern blotting, to measure short telomeres in individual cells. The mean short telomere length measured in individual cells by USC-STELA correlates with that from bulk cells, measured by Universal STELA (U-STELA). The validation and reproducibility of USC-STELA was confirmed using different cell types with known telomere lengths, as well as by using paired sister-cells from human embryos and cultured cells. Interestingly, individual cells known to elongate telomeres via alternative lengthening of telomeres (ALT) have more short telomeres, yet longer mean telomere length than control cells. Moreover, individual senescent fibroblasts carry more short telomeres compared to human embryonic stem cells (hESCs), consistent with the notion that short telomeres contribute to cellular senescence. Additionally, we found a greater load of short telomeres in polar bodies than in matching oocytes, providing further insights into the accelerated polar body DNA degradation following extrusion from the oocyte. USC-STELA provides a new method to study telomere dysfunction in individual cells, with potential to improve our understanding of the role of telomere dynamics in cancer, developmental biology and reproductive medicine.
Paula Andrea Navarro,
Xinghua Victor Pan,
Measurement of Short Telomere Load in Individual Cells, International Journal of Genetics and Genomics.
Vol. 7, No. 3,
2019, pp. 60-68.
Griffith JD, Comeau L, Rosenfield S, Stansel RM, Bianchi A, Moss H, de Lange T: Mammalian telomeres end in a large duplex loop. Cell 1999, 97 (4): 503-514.
de Lange T: Shelterin: the protein complex that shapes and safeguards human telomeres. Genes & development 2005, 19 (18): 2100-2110.
Olovnikov AM: A theory of marginotomy. The incomplete copying of template margin in enzymic synthesis of polynucleotides and biological significance of the phenomenon. J Theor Biol 1973, 41 (1): 181-190.
Levy MZ, Allsopp RC, Futcher AB, Greider CW, Harley CB: Telomere end-replication problem and cell aging. J Mol Biol 1992, 225 (4): 951-960.
Von Zglinicki T: Oxidative stress shortens telomeres. Trends Biochem Sci 2002, 27 (7): 339-344.
Epel ES, Blackburn EH, Lin J, Dhabhar FS, Adler NE, Morrow JD, Cawthon RM: Accelerated telomere shortening in response to life stress. Proc Natl Acad Sci U S A 2004, 101 (49): 17312-17315.
Denham J, Nelson CP, O'Brien BJ, Nankervis SA, Denniff M, Harvey JT, Marques FZ, Codd V, Zukowska-Szczechowska E, Samani NJ et al: Longer leukocyte telomeres are associated with ultra-endurance exercise independent of cardiovascular risk factors. PLoS One 2013, 8 (7): e69377.
Zota AR, Needham BL, Blackburn EH, Lin J, Park SK, Rehkopf DH, Epel ES: Associations of cadmium and lead exposure with leukocyte telomere length: findings from National Health and Nutrition Examination Survey, 1999-2002. Am J Epidemiol 2015, 181 (2): 127-136.
Abdallah P, Luciano P, Runge KW, Lisby M, Geli V, Gilson E, Teixeira MT: A two-step model for senescence triggered by a single critically short telomere. Nat Cell Biol 2009, 11 (8): 988-993.
Harley CB, Futcher AB, Greider CW: Telomeres shorten during ageing of human fibroblasts. Nature 1990, 345 (6274): 458-460.
Slijepcevic P: Telomere length measurement by Q-FISH. Methods Cell Sci 2001, 23 (1-3): 17-22.
Kimura M, Stone RC, Hunt SC, Skurnick J, Lu X, Cao X, Harley CB, Aviv A: Measurement of telomere length by the Southern blot analysis of terminal restriction fragment lengths. Nat Protoc 2010, 5 (9): 1596-1607.
Bendix L, Horn PB, Jensen UB, Rubelj I, Kolvraa S: The load of short telomeres, estimated by a new method, Universal STELA, correlates with number of senescent cells. Aging Cell 2010, 9 (3): 383-397.
Xu Z, Duc KD, Holcman D, Teixeira MT: The length of the shortest telomere as the major determinant of the onset of replicative senescence. Genetics 2013, 194 (4): 847-857.
Wang F, Pan X, Kalmbach K, Seth-Smith ML, Ye X, Antumes DM, Yin Y, Liu L, Keefe DL, Weissman SM: Robust measurement of telomere length in single cells. Proceedings of the National Academy of Sciences of the United States of America 2013, 110 (21): E1906-1912.
Smallwood SA, Lee HJ, Angermueller C, Krueger F, Saadeh H, Peat J, Andrews SR, Stegle O, Reik W, Kelsey G: Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity. Nature methods 2014, 11 (8): 817-820.
Buettner F, Natarajan KN, Casale FP, Proserpio V, Scialdone A, Theis FJ, Teichmann SA, Marioni JC, Stegle O: Computational analysis of cell-to-cell heterogeneity in single-cell RNA-sequencing data reveals hidden subpopulations of cells. Nature biotechnology 2015, 33 (2): 155-160.
Huang L, Ma F, Chapman A, Lu S, Xie XS: Single-Cell Whole-Genome Amplification and Sequencing: Methodology and Applications. Annu Rev Genomics Hum Genet 2015, 16: 79-102.
Guo H, Zhu P, Wu X, Li X, Wen L, Tang F: Single-cell methylome landscapes of mouse embryonic stem cells and early embryos analyzed using reduced representation bisulfite sequencing. Genome Res 2013, 23 (12): 2126-2135.
Collerton J, Martin-Ruiz C, Kenny A, Barrass K, von Zglinicki T, Kirkwood T, Keavney B, Newcastle 85+ Core Study T: Telomere length is associated with left ventricular function in the oldest old: the Newcastle 85+ study. European heart journal 2007, 28 (2): 172-176.
Henson JD, Reddel RR: Assaying and investigating Alternative Lengthening of Telomeres activity in human cells and cancers. FEBS Lett 2010, 584 (17): 3800-3811.
Rajaraman S, Choi J, Cheung P, Beaudry V, Moore H, Artandi SE: Telomere uncapping in progenitor cells with critical telomere shortening is coupled to S-phase progression in vivo. Proc Natl Acad Sci U S A 2007, 104 (45): 17747-17752.
d'Adda di Fagagna F: Living on a break: cellular senescence as a DNA-damage response. Nat Rev Cancer 2008, 8 (7): 512-522.
Treff NR, Su J, Taylor D, Scott RT, Jr.: Telomere DNA deficiency is associated with development of human embryonic aneuploidy. PLoS genetics 2011, 7 (6): e1002161.
Voytas D: Agarose gel electrophoresis. Curr Protoc Mol Biol 2001, Chapter 2: Unit2 5A.
Cha RS, Thilly WG: Specificity, efficiency, and fidelity of PCR. PCR Methods Appl 1993, 3 (3): S18-29.