24ASSESSMENT OF TELOMERE LENGTH IN NUCLEAR TRANSFER DERIVED SHEEP CLONES, THEIR OFFSPRING, AND CONTROL ANIMALS

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 (2mg) were digested at 37°C for 2h 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 0.1s switch time for 4h 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 10–19kb. 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 0.08kb 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 11.12kb, 11.40kb, and 13.41kb, respectively. Their mean TRF values were significantly (P<0.05) shorter than those of their age-matched control animals: 16.95kb, 14.76kb, and 14.76kb, 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±0.69kb v. 16.28±0.72kb). 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.)