PARENT SESSION
Aging

ASSESSMENT OF TELOMERE LENGTH IN CLONED GOATS.

Betts, Dean¹, Favetta, Laura¹, Perrault, Steven¹, Keefer, Carol², King, Allan¹, ^{1 2}

ABSTRACT- Successful animal cloning from somatic cells requires the reversal of cellular differentiation and aging events to restore the genome to a totipotent, youthful state. Recent studies evaluating the telomere length status of cloned bovine and ovine offspring have demonstrated conflicting results. The aims of this study were to measure and compare the telomere lengths of cloned goats, the somatic donor cell cultures and control animals. Two cloned male goats (1 yr) and their younger clone (3 months old) derived from fetal fibroblasts (FF), six cloned females (2-3 months old) derived from adult, granulosa cells (GC), donor cell lines and controls were analyzed. Isolated genomic DNA samples (2.5-10 g) from ear biopsies were digested using a Hinf I/Rsa I enzyme mixture and the mean telomere lengths were determined by telomere restriction fragment (TRF) analysis using the TeloTAGGG Telomere Length Assay kit (Roche Molecular Biochemicals). As in other species, telomere shortening was observed during in vitro culture of goat fetal fibroblast and granulosa cell cultures. However, unlike the situation in cattle, telomere lengths were significantly (P<0.05) shorter in the FF clones (16.96 ± 1.64 kb) and CG clones (18.77 ± 0.67 kb) than control animals at 1 year of age (20.48 kb). There was high variability in telomere lengths among individual clones derived from the same donor cell line at the same passage for FF (13.83-19.39 kb) and GC (17.41-20.01 kb) clones. One of the 1-year old FF clones exhibited shorter mean TRF lengths (13.83 kb) than a 3- to 4-year old control male (16.97 kb). These results suggest that cloned goats do not fully rebuild their telomeres. The discrepancies in telomere restoration reported on cloned animals could be due to differences between species, donor cell types, nuclear transfer procedures, and other regulatory mechanisms, such as the expression of telomere-specific binding proteins. The telomere length variations between cloned goats of the same age could arise from donor cell selection, and/or animal-specific differences in telomere restoration and cellular proliferation. (Research supported by the Canadian Institutes of Health Research (CIHR), Natural Sciences and Engineering Council of Canada (NSERC) and Nexia Biotechnologies Inc.)

KEY WORDS: nuclear transfer, telomere length, reprogramming, goat