33 TELOMERASE ACTIVITIES IN CLONED BEAGLE DOGS

2015 ◽  
Vol 27 (1) ◽  
pp. 109
Author(s):  
G. A. Kim ◽  
H. J. Oh ◽  
M. J. Kim ◽  
Y. K. Jo ◽  
E. M. N. Setyawan ◽  
...  
Keyword(s):  
Telomere Length ◽  
Donor Cell ◽  
Telomerase Activity ◽  
Physiological Age ◽  
Biological Aging ◽  
Amplification Protocol ◽  
Donor Cells ◽  
Activity Measurements ◽  
Natural Breeding

Telomerase is important ribonucleoprotein for restoring telomere length from its own RNA template. Regarding cloned animals derived from somatic cell nuclear transfer (SCNT), interesting questions have been raised about whether the cloning process restores cellular telomerase activity undergone by their donor cells. The present study was conducted to determine the effects of cloning on telomerase activity in the dog and normality of telomerase activity in cloned dogs. Focusing our attention on differences in telomerase activity depending on the age, we analysed telomerase activity in dogs produced by natural breeding of various ages. Comparison of the telomerase activities of cloned dogs and those of dogs produced by natural breeding was also performed. For SCNT, 2 cell donors, 7- and 9-year-old beagles, were used and donor cells were isolated from ear skin. After establishing donor cell lines, the enucleated canine in vivo-matured oocytes and the cells were injected and fused by electrofusion. After 30 days from embryo transfer, pregnancy diagnosis was performed and 7 cloned dogs were produced on the due date. For standardization of telomerase activity in beagles produced by natural breeding, blood of total 14 dogs at each age (10 months, 20 months, 5, 7, and 8 years old) were collected and telomerase activity was measured by the telomeric repeat amplification protocol (TRAP) assay. Telomerase activity measurements of at least 6 replications in each dog were performed. For statistical analysis, one-way ANOVA with Dunn's Multiple Comparison Test was used. Significant differences in telomerase activity were observed between the blood of cloned and donor dogs. It was shown that mean telomerase activities were decreased according to biological aging with significances. Mean telomerase activities in 10 cloned dogs were higher than those of a donor dog. Cloned dogs also showed similar levels of telomerase activities as their age-matched natural bred dogs, suggesting that they are within the variation in normal dogs. These observations indicate that the cloning process restores the telomerase activity in the dog. Thus, complex regulation of telomerase activity during nuclear reprogramming may regulate and be involved in telomerase activity in cloned dogs. It remains to be determined whether telomere length is correlated with telomerase activity and if it accurately reflects the physiological age of cloned dogs.This study was supported by IPET (#311062–04–2-SB010), RDA (PJ008975022013), Research Institute for Veterinary Science, the BK21 program, Nestle Purina Korea, and TS Corporation.

scholarly journals Dynamic integration of enteric neural stem cells in ex vivo organotypic colon cultures

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Georgina Navoly ◽  
Conor J. McCann
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Organotypic Culture ◽  
Ex Vivo ◽  
Donor Cell ◽  
Colonic Tissue ◽  
Cell Therapies ◽  
Enteric Ganglia ◽  
Donor Cells

AbstractEnteric neural stem cells (ENSC) have been identified as a possible treatment for enteric neuropathies. After in vivo transplantation, ENSC and their derivatives have been shown to engraft within colonic tissue, migrate and populate endogenous ganglia, and functionally integrate with the enteric nervous system. However, the mechanisms underlying the integration of donor ENSC, in recipient tissues, remain unclear. Therefore, we aimed to examine ENSC integration using an adapted ex vivo organotypic culture system. Donor ENSC were obtained from Wnt1cre/+;R26RYFP/YFP mice allowing specific labelling, selection and fate-mapping of cells. YFP+ neurospheres were transplanted to C57BL6/J (6–8-week-old) colonic tissue and maintained in organotypic culture for up to 21 days. We analysed and quantified donor cell integration within recipient tissues at 7, 14 and 21 days, along with assessing the structural and molecular consequences of ENSC integration. We found that organotypically cultured tissues were well preserved up to 21-days in ex vivo culture, which allowed for assessment of donor cell integration after transplantation. Donor ENSC-derived cells integrated across the colonic wall in a dynamic fashion, across a three-week period. Following transplantation, donor cells displayed two integrative patterns; longitudinal migration and medial invasion which allowed donor cells to populate colonic tissue. Moreover, significant remodelling of the intestinal ECM and musculature occurred upon transplantation, to facilitate donor cell integration within endogenous enteric ganglia. These results provide critical evidence on the timescale and mechanisms, which regulate donor ENSC integration, within recipient gut tissue, which are important considerations in the future clinical translation of stem cell therapies for enteric disease.

scholarly journals Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation

Reproduction ◽  
2007 ◽  
Vol 133 (1) ◽  
pp. 219-230 ◽  
Author(s):  
Feikun Yang ◽  
Ru Hao ◽  
Barbara Kessler ◽  
Gottfried Brem ◽  
Eckhard Wolf ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Epigenetic Mark ◽  
Cell Type ◽  
Developmental Potential ◽  
Acetylation Status ◽  
Donor Cells ◽  
Donor Cell Type

The epigenetic status of a donor nucleus has an important effect on the developmental potential of embryos produced by somatic cell nuclear transfer (SCNT). In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Alicia/Basilea) into metaphase II oocytes and analyzed the levels of histone H3-lysine 9-lysine 14 acetylation (acH3K9/14) in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with blastomeres fromin vivofertilized or parthenogenetic embryos. The levels of acH3K9/14 were higher in RCCs than in RFFs (P<0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC cloned embryos induced a higher initial pregnancy rate as compared to RFF cloned embryos (40 vs 20%). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed, live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly increased the level of acH3K9/14 and the proportion of nuclear transfer embryos developing to blastocyst (49 vs 33% with non-treated RFF,P<0.05). The distribution of acH3K9/14 in either group of cloned embryos did not resemble that inin vivofertilized embryos suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres fromin vivoderived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and may be a useful epigenetic mark to predict efficiency of SCNT in rabbits.

scholarly journals Telomere Length in Valve Tissue Is Shorter in Individuals With Aortic Stenosis and in Calcified Valve Areas

2021 ◽  
Vol 9 ◽  
Author(s):  
Ilona Saraieva ◽  
Athanase Benetos ◽  
Carlos Labat ◽  
Anders Franco-Cereceda ◽  
Magnus Bäck ◽  
...  
Keyword(s):  
Aortic Valve ◽  
Aortic Stenosis ◽  
Telomere Length ◽  
Telomerase Activity ◽  
Aortic Valves ◽  
Fragment Analysis ◽  
Replacement Surgery ◽  
Age Related ◽  
Amplification Protocol ◽  
Telomere Restriction Fragment

BackgroundShort telomere length (TL) is associated with age-related diseases, in particular cardiovascular diseases. However, whether the onset and course of aortic stenosis (AS) is linked to TL in aortic valves remains unknown.ObjectivesTo assess telomere dynamics (TL and telomerase activity) in aortic valves and the possible implication of TL in onset and course of AS.MethodsDNA was extracted from aortic valves obtained from 55 patients (78.2% men; age, 37–79 years), who had undergone replacement surgery due to AS (AS group, n = 32), aortic valve regurgitation and aortic dilation (Non-AS group, n = 23). TL was measured by telomere restriction fragment analysis (TRF) in calcified and non-calcified aortic valve areas. Telomerase activity was evaluated using telomerase repeat amplification protocol (TRAP) in protein extracts from non-calcified and calcified areas of valves obtained from 4 additional patients (50% men; age, 27–70 years).ResultsTL was shorter in calcified aortic valve areas in comparison to non-calcified areas (n = 31, 8.58 ± 0.73 kb vs. 8.12 ± 0.75 kb, p &lt; 0.0001), whereas telomerase activity was not detected in any of those areas. Moreover, patients from AS group displayed shorter telomeres in non-calcified areas than those from the Non-AS group (8.40 ± 0.64 kb vs. 8.85 ± 0.65, p = 0.01).ConclusionsShort telomeres in aortic valves may participate in the development of AS, while concurrently the calcification process seems to promote further local decrease of TL in calcified areas of valves.

scholarly journals TIN2 Functions with TPP1/POT1 To Stimulate Telomerase Processivity

2019 ◽  
Vol 39 (21) ◽  
Author(s):  
Alexandra M. Pike ◽  
Margaret A. Strong ◽  
John Paul T. Ouyang ◽  
Carol W. Greider
Keyword(s):  
Telomere Length ◽  
Telomerase Activity ◽  
Human Cells ◽  
Short Telomere ◽  
Gene Encoding ◽  
Important Regulator

ABSTRACT TIN2 is an important regulator of telomere length, and mutations in TINF2, the gene encoding TIN2, cause short-telomere syndromes. While the genetics underscore the importance of TIN2, the mechanism through which TIN2 regulates telomere length remains unclear. Here, we tested the effects of human TIN2 on telomerase activity. We identified a new isoform in human cells, TIN2M, that is expressed at levels similar to those of previously studied TIN2 isoforms. All three TIN2 isoforms localized to and maintained telomere integrity in vivo, and localization was not disrupted by telomere syndrome mutations. Using direct telomerase activity assays, we discovered that TIN2 stimulated telomerase processivity in vitro. All of the TIN2 isoforms stimulated telomerase to similar extents. Mutations in the TPP1 TEL patch abrogated this stimulation, suggesting that TIN2 functions with TPP1/POT1 to stimulate telomerase processivity. We conclude from our data and previously published work that TIN2/TPP1/POT1 is a functional shelterin subcomplex.

scholarly journals Yeast Est2p Affects Telomere Length by Influencing Association of Rap1p with Telomeric Chromatin

2008 ◽  
Vol 28 (7) ◽  
pp. 2380-2390 ◽  
Author(s):  
Hong Ji ◽  
Christopher J. Adkins ◽  
Bethany R. Cartwright ◽  
Katherine L. Friedman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Telomere Length ◽  
Specific Binding ◽  
Telomerase Activity ◽  
Negative Regulator ◽  
Wild Type ◽  
Telomeric Dna ◽  
Telomere Length Homeostasis

ABSTRACT In Saccharomyces cerevisiae, the sequence-specific binding of the negative regulator Rap1p provides a mechanism to measure telomere length: as the telomere length increases, the binding of additional Rap1p inhibits telomerase activity in cis. We provide evidence that the association of Rap1p with telomeric DNA in vivo occurs in part by sequence-independent mechanisms. Specific mutations in EST2 (est2-LT) reduce the association of Rap1p with telomeric DNA in vivo. As a result, telomeres are abnormally long yet bind an amount of Rap1p equivalent to that observed at wild-type telomeres. This behavior contrasts with that of a second mutation in EST2 (est2-up34) that increases bound Rap1p as expected for a strain with long telomeres. Telomere sequences are subtly altered in est2-LT strains, but similar changes in est2-up34 telomeres suggest that sequence abnormalities are a consequence, not a cause, of overelongation. Indeed, est2-LT telomeres bind Rap1p indistinguishably from the wild type in vitro. Taken together, these results suggest that Est2p can directly or indirectly influence the binding of Rap1p to telomeric DNA, implicating telomerase in roles both upstream and downstream of Rap1p in telomere length homeostasis.

Telomerase Activity and Telomere Length of CD4+CD25+ Regulatory T-Cells under Conditions of In Vitro and In Vivo Expansion.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3857-3857
Author(s):  
Dominik G.F. Wolf ◽  
Anna M. Wolf ◽  
Christian Koppelstaetter ◽  
Holger F. Rumpold ◽  
Gert Mayer ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Telomere Length ◽  
Cancer Patients ◽  
Telomerase Activity ◽  
Telomere Maintenance ◽  
Healthy Human ◽  
Telomere Shortening

Abstract The expandability of CD4+CD25+ regulatory T-cells (Treg) has been shown in vitro and in vivo. Activation of telomerase activity is a prerequisite for clonal expansion and telomere maintenance in T-cells. There is currently no data available on the expression and function of telomerase in proliferating Treg. Analyses of telomere length by flow-FISH, real-time PCR and Southern blotting revealed that Treg isolated from healthy human volunteers have significantly shortened telomeres when compared to CD4+CD25− T-cells. However, telomere length is not further shortened in Treg isolated from the peripheral blood of cancer patients, despite the observation that the regulatory T-cell pool of these patients was significantly enlarged. To gain further insight into maintenance of telomere length of Treg, we induced in vitro proliferation of Treg by stimulation with anti-CD3 and IL-2. This led to a rapid increase of telomerase activity, as determined by PCR-ELISA. However, when we focused on the proliferating fraction of Treg using a sorting strategy based on the dilution of CFSE, we could show a significant telomere shortening in Treg with high proliferative and immmuno-suppressive capacity. Of note, proliferating CFSElow Treg are characterized by high telomerase activity, which however seems to be insufficient to avoid further telomere shortening under conditions of strong in vitro stimulation. In contrast, under conditions of in vivo expansion of Treg in cancer patients, the induction of telomerase activity is likely to compensate for further telomere erosion. These data might be of importance when considering the application of in vitro expanded Treg for the treatment of GvHD or autoimmune diseases, as telomere shortening might be associated with genomic instability.

GERD is associated with shortened telomeres in the squamous epithelium of the distal esophagus

2007 ◽  
Vol 293 (1) ◽  
pp. G19-G24 ◽  
Author(s):  
Rhonda F. Souza ◽  
Tisha Lunsford ◽  
Ruben D. Ramirez ◽  
Xi Zhang ◽  
Edward L. Lee ◽  
...  
Keyword(s):  
Telomere Length ◽  
Barrett’S Esophagus ◽  
Dna Sequences ◽  
Barrett's Esophagus ◽  
Oxidative Dna Damage ◽  
Squamous Epithelium ◽  
Telomerase Activity ◽  
Mucosal Healing ◽  
Distal Esophagus ◽  
Amplification Protocol

Telomeres are repetitive DNA sequences located at the ends of chromosomes. Telomeres are shortened by repeated cell divisions and by oxidative DNA damage, and cells with critically shortened telomeres cannot divide. We hypothesized that chronic gastroesophageal reflux disease (GERD)-induced injury of the esophageal squamous epithelium results in progressive telomeric shortening that eventually might interfere with mucosal healing. To address our hypothesis, we compared telomere length and telomerase activity in biopsy specimens of esophageal squamous epithelium from GERD patients and control patients. Endoscopic biopsies were taken from the esophageal squamous epithelium of 38 patients with GERD [10 long-segment Barrett's esophagus (LSBE), 15 short-segment (SSBE), 13 GERD without Barrett's esophagus] and 16 control patients without GERD. Telomere length was assessed using the terminal restriction fragment assay, and telomerase activity was studied by the PCR-based telomeric repeat amplification protocol assay. Patients with GERD had significantly shorter telomeres in the distal esophagus than controls [8.3 ± 0.5 vs. 10.9 ± 1.5 (SE) Kbp, P = 0.043]. Among the patients with GERD, telomere length in the distal esophagus did not differ significantly in those with and without Barrett's esophagus (LSBE 7.9 ± 0.8, SSBE 8.6 ± 0.9, GERD without BE 8.7 ± 1.0 Kbp). No significant differences in telomerase activity in the distal esophagus were noted between patients with GERD and controls (4.0 ± 0.39 vs. 5.2 ± 0.53 RIUs). Telomeres in the squamous epithelium of the distal esophagus of patients who have GERD, with and without Barrett's esophagus, are significantly shorter than those of patients without GERD despite similar levels of telomerase activity.

scholarly journals Vault Poly(ADP-Ribose) Polymerase Is Associated with Mammalian Telomerase and Is Dispensable for Telomerase Function and Vault Structure In Vivo

2004 ◽  
Vol 24 (12) ◽  
pp. 5314-5323 ◽  
Author(s):  
Yie Liu ◽  
Bryan E. Snow ◽  
Valerie A. Kickhoefer ◽  
Natalie Erdmann ◽  
Wen Zhou ◽  
...  
Keyword(s):  
Telomere Length ◽  
Telomerase Activity ◽  
Molecular Assembly ◽  
Cellular Functions ◽  
Telomere Function ◽  
Minor Protein ◽  
The Stability ◽  
A Minor

ABSTRACT Vault poly(ADP-ribose) polymerase (VPARP) was originally identified as a minor protein component of the vault ribonucleoprotein particle, which may be involved in molecular assembly or subcellular transport. In addition to the association of VPARP with the cytoplasmic vault particle, subpopulations of VPARP localize to the nucleus and the mitotic spindle, indicating that VPARP may have other cellular functions. We found that VPARP was associated with telomerase activity and interacted with exogenously expressed telomerase-associated protein 1 (TEP1) in human cells. To study the possible role of VPARP in telomerase and vault complexes in vivo, mVparp-deficient mice were generated. Mice deficient in mVparp were viable and fertile for up to five generations, with no apparent changes in telomerase activity or telomere length. Vaults purified from mVparp-deficient mouse liver appeared intact, and no defect in association with other vault components was observed. Mice deficient in mTep1, whose disruption alone does not affect telomere function but does affect the stability of vault RNA, showed no additional telomerase or telomere-related phenotypes when the mTep1 deficiency was combined with an mVparp deficiency. These data suggest that murine mTep1 and mVparp, alone or in combination, are dispensable for normal development, telomerase catalysis, telomere length maintenance, and vault structure in vivo.

scholarly journals Telomerase-Associated Protein TEP1 Is Not Essential for Telomerase Activity or Telomere Length Maintenance In Vivo

2000 ◽  
Vol 20 (21) ◽  
pp. 8178-8184 ◽  
Author(s):  
Yie Liu ◽  
Bryan E. Snow ◽  
M. Prakash Hande ◽  
Gabriela Baerlocher ◽  
Valerie A. Kickhoefer ◽  
...  
Keyword(s):  
Telomere Length ◽  
Rna Binding ◽  
Es Cells ◽  
Embryonic Stem ◽  
Telomerase Activity ◽  
Telomerase Rna ◽  
Wild Type ◽  
Rnp Complex ◽  
Successive Generations

ABSTRACT TEP1 is a mammalian telomerase-associated protein with similarity to the Tetrahymena telomerase protein p80. Like p80, TEP1 is associated with telomerase activity and the telomerase reverse transcriptase, and it specifically interacts with the telomerase RNA. To determine the role of mTep1 in telomerase function in vivo, we generated mouse embryonic stem (ES) cells and mice lacking mTep1. ThemTep1-deficient (mTep1 −/−) mice were viable and were bred for seven successive generations with no obvious phenotypic abnormalities. All murine tissues frommTep1 −/− mice possessed a level of telomerase activity comparable to that in wild-type mice. In addition, analysis of several tissues that normally lack telomerase activity revealed no reactivation of telomerase activity in mTep1 −/− mice. Telomere length, even in later generations ofmTep1 −/− mice, was equivalent to that in wild-type animals. ES cells deficient in mTep1 also showed no detectable alteration in telomerase activity or telomere length with increased passage in culture. Thus, mTep1 appears to be completely dispensable for telomerase function in vivo. Recently, TEP1 has been identified within a second ribonucleoprotein (RNP) complex, the vault particle. TEP1 can also specifically bind to a small RNA, vRNA, which is associated with the vault particle and is unrelated in sequence to mammalian telomerase RNA. These results reveal that TEP1 is an RNA binding protein that is not restricted to the telomerase complex and that TEP1 plays a redundant role in the assembly or localization of the telomerase RNP in vivo.

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