Quantitative Analysis of Telomerase Activity and Telomere Length in Domestic Animal Clones

  • Dean H. Betts
  • Steven Perrault
  • Lea Harrington
  • W. Allan King
Part of the Methods in Molecular Biology™ book series (MIMB, volume 325)

Abstract

It has been speculated that incomplete epigenetic reprogramming of the somatic cell genome is the primary reason behind the developmental inefficiencies and postnatal abnormalities observed after nuclear transplantation in domestic animal clones. One chromosome structure that is altered in dividing somatic cells is telomere length—the terminal ends of linear chromosomes capped by repetitive sequences of G-rich noncoding DNA, (TTAGGG)n, and specific binding proteins. Telomeres are critical structures that function in maintaining chromosome stability and ensure the full replication of coding DNA by acting as a buffer to terminal DNA attrition due to the end replication problem. Telomere shortening limits cellular proliferation through a DNA damage signal activating permanent cell cycle arrest at a critical telomere length or through structural telomere alterations that prevents effective chromosome capping. Telomere-mediated signaling of cellular senescence has been established for many somatic cell types in vitro, except for germ cells, cancer lines, and regenerative tissues in which telomere length is maintained primarily by the ribonucleoprotein telomerase, a reverse transcriptase that synthesizes TTAGGG repeats de novo onto the chromosome ends. Telomere length discrepancies have been reported in animal clones as being shorter, no different, and even longer than in age-matched control animals, but the etiology is not yet understood. Possible explanations include differences in donor cell type and the efficiency of telomerase reprogramming. This chapter summarizes the conventional protocols and recent advances in telomere length and telomerase activity measurement that will help elucidate the mechanism(s) behind telomere length deregulation in somatic cell clones and its role in chromosomal instability, cellular senescence, and organismal aging in vivo.

Key Words

Telomere TRF Q-FISH telomerase TRAP RQ-TRAP animal cloning SCNT aging 

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Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Dean H. Betts
    • 1
  • Steven Perrault
    • 1
  • Lea Harrington
    • 2
    • 3
  • W. Allan King
    • 1
  1. 1.Department of Biomedical Sciences, Ontario Veterinary CollegeUniversity of GuelphGuelphCanada
  2. 2.The Department of Medical BiophysicsThe University of TorontoToronto
  3. 3.Princess Margaret HospitalTorontoCanada

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