48 THE AMOUNT OF TELOMERIC DNA IN LYMPHOCYTES OF DOG CLONES

Abstract

Controversy regarding the restoration of eroded telomere length of donor cells after the nuclear transfer process has arisen from previous studies of cloned cattle, mice, and pigs. Little is known about telomere lengths in dogs from either natural breeding or somatic cell nuclear transfer (SCNT). In this study, we investigated the amount of telomeric DNA (ATD) in the lymphocytes of growing dog clones and their somatic cell donors. One cloned male Afghan hound dog [Snuppy (Lee et al. 2005 Nature 436, 641)] and 3 cloned female Afghan hound dogs (Jang et al. 2006 Theriogenology; doi:10.1016J.THERIOGENOLOGY.2006.11.006) were obtained from somatic cell nuclear transfer (SCNT) of ear skin fibroblasts. The lymphocytes were recovered from all dog clones: their nuclear donor dogs (male donor dog, female donor dog), and six normal Afghan hound dogs (control, and 10-, 20-, 26-, 49-, 55-, and 58-month-old, respectively). The ATD was analyzed by quantitative fluorescence in situ hybridization (Q-FISH) with a telomeric DNA probe. A telomeric probe containing the TTAGGG repeated DNA sequence was simultaneously amplified and labeled with digoxigenin (DIG) by polymerase chain reaction (PCR) using dog genomic DNA as template, a (GGGTAA)7 primer, and a DIG-labeling kit (Roche, Mannheim, Germany). To analyze the amount of telomeric DNA on the lymphocytes, at least 100 interphase nuclei were examined in each specimen. The image was captured by a digital camera (DP-70, Olympus) and analyzed using MetaMorph (Universal Imaging Co., Downingtown, PA, USA), an image analysis program. Our results indicated that the ATD in normal Afghan hounds gradually decreased with age. Although no difference in ATD was observed between 10- and 26-month-old dogs, the ATD in the 26-month-old dog was significantly higher than that in 49-, 55-, and 58-month-olds (P < 0.05). The mean percentage of telomeric DNA in Snuppy (18-month-old; 2.38%) was significantly higher than that in the nuclear donor dog (49-month-old; 2.12%) but less than that in the age-matched control (20-month-old; 2.72%; P < 0.05). The ATD in 3 female clone dogs (3-, 2-, and 2-month-olds; 3.47, 3.28, and 3.07%) were significantly higher than that in the nuclear donor (26-month-old; 2.65%). In conclusion, the mean percentages of telomeric DNA in dog clones were higher than in nuclear donor dogs, and the ATD of the cloned male dog was different from that in age-matched controls from natural reproduction. The results suggest that the amount of telomeric DNA in dog clones can be restored with the nuclear transfer of cultured donor fibroblasts, but further studies are required as to how telomere reprogramming occurs during the nuclear transfer process.