24 ASSESSMENT OF TELOMERE LENGTH IN NUCLEAR TRANSFER DERIVED SHEEP CLONES, THEIR OFFSPRING, AND CONTROL ANIMALS
Bibliographic record
Abstract
The technique of somatic cell nuclear transfer (SCNT) involves the transplantation of a cultured somatic cell into an enucleated oocyte. Following activation, the nucleus of the somatic cell should be reprogrammed to a state of totipotency, which involves the resetting of epigenetic modifications of the somatic genome and also rebuilding of the chromosomal ends (telomeres). This study was carried out to investigate whether the nuclear transfer technique could rebuild the telomeres in sheep clones and their offspring when compared to age-matched control animals. Skin and blood samples were collected from three SCNT-derived sheep clones, and three of their offspring generated by natural mating. Control samples were collected from age-matched animals (n = 17), spanning an age range from 1 month to 36 months of age. Ovine genomic DNA was extracted from samples using a Qiagen DNA extraction kit according to the manufacturer’s instructions. TeloTAGGG telomere length chemiluminescent assay kit (Roche Diagnostics) was used to determine the telomere length of all samples. Samples of ovine genomic DNA (2 mg) were digested at 37°C for 2 h using Hinf 1 and Rsa 1 endonuclease master-mix at the rate of 40 IU per sample. The resulted terminal restriction fragments (TRF) were separated on 1.2% agarose gel by pulsed field electrophoresis at 6V with 0.1 s switch time for 4 h in 0.5% TBE buffer at 14°C. The DNA fragments were southern-transferred to a nylon membrane and hybridized with telomere-specific (TTAGGG)n, digoxigenin (DIG)-labeled hybridization probe at 42°C. Following post-hybridization washes the membrane was incubated with DIG-specific antibody covalently coupled to alkaline phosphatase. The immobilized telomere probe was visualized by adding a chemiluminecence substrate to the membrane and exposed to X-ray film. Mean TRF length was determined by comparing the telomere densitometry signals to a molecular weight standard. High resolution bands resulting from pulsed field gel electrophoresis revealed that ovine TRF size distribution was in the range of 10–19 kb. Linear regression analysis resulted in significant (t = 25.84, P < 0.05) decrease of mean TRF size with increasing age, at a mean rate of 0.08 kb per every 6 months. Mean TRF length of SCNT-derived sheep clones, Matilda (28 months old), Macather (14 months old) and Frida (13 months old) were 11.12 kb, 11.40 kb, and 13.41 kb, respectively. Their mean TRF values were significantly (P < 0.05) shorter than those of their age-matched control animals: 16.95 kb, 14.76 kb, and 14.76 kb, respectively (n = 3 per group). The mean TRF length of the 3 offspring (21 months old) derived from natural mating was not significantly (P < 0.05) different from age-matched controls (17.75 ± 0.69 kb v. 16.28 ± 0.72 kb). These results demonstrate that sheep clones derived from cultured somatic cells have shorter telomere lengths compared to age-matched controls, but this telomere loss is reset in the subsequent generation through natural breeding of the clones. (Funded by NSERC, OMAFRA and International Council for Canadian Studies.)
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".