34 COMPARISON OF PROLIFERATION AND TELOMERASE ACTIVITY IN FIBROBLASTS DERIVED FROM GYEONG-JU DONGGYEONG DOGS ACCORDING TO THEIR AGE

Abstract

Donggyeong dog is a breed considered a natural monument in Korea since 2012. Nevertheless, this breed is only found in Gyeong-ju and is classified as endangered. The use of assisted reproductive technologies (ART) could be applied to preserve these endangered dogs. Among various ART, somatic cell nuclear transfer (SCNT) may be a good technique for achieving propagation of genetically identical individuals. For this reason, we investigated the effect of age of the cell donor on several characteristics, including growth pattern, doubling time of cell populations, cell size, viability, and telomerase activity in Gyeong-ju Donggyeong dog fibroblast cultures before SCNT. Primary fibroblast cell cultures were performed using ear biopsies from 1-year-old (D-1yr) and 7-year-old (D-7yr) Donggyeong dogs and cells at passage 2 to 6 from in this study. Cells were plated at 1 × 10⁵ cells well^–1 in a 6-well plate. Cells were harvested every 24 h for 6 days, and cell number was determined to measure growth pattern and doubling time. The harvested cells were stained with trypan blue, their size and viability were analysed using a Countess Automated Cell Counter (Life Technologies, Carlsbad, CA, USA). Telomerase activity was measured by telomeric repeat amplification protocol using TeloTAGGG Telomerase PCR ELISAPLUS. All experiments were replicated at least 3 times. Statistical analysis was performed using Graphpad Prism (GraphPad Software Inc., San Diego, CA, USA), and t-test (P < 0.05) was used to compare the growth pattern, doubling time, cell size, viability, and telomerase activity between D-1yr and D-7yr groups. Growth curves in both groups showed the typical “S” shape and no significant differences between D-1yr and D-7yr groups. However, doubling times from 2nd to 5th passages of D-1yr (20.0 ± 0.3 h, 38.0 ± 0.5 h, 38.7 ± 0.5 h, and 53.4 ± 1.4 h) were significantly shorter than those of D-7yr (37.0 ± 1.2 h, 54.0 ± 3.6 h, 63.3 ± 1.8 h, and 100.9 ± 4.4 h). Cells from 6th passage of D-7yr and 7th passage of D-1yr did not reach confluence. Cell size of D-1yr (13.6 ± 0.2 μm) was significantly smaller than that of D-7yr (14.9 ± 0.3 μm; P < 0.001). There was no significant difference between D-1yr (89.4 ± 1.4%) and D-7yr (88.9 ± 1.2%) in cell viability but relative telomerase activity was significantly higher in D-1yr (37.8 ± 7.5) compared with D-7yr (19.0 ± 6.2, P < 0.001). In conclusion, these results suggest that fibroblasts derived from young Donggyeong dogs are more suitable for SCNT than those from old donors. For further study, dog cloning using D-1yr and D-7yr should be performed to evaluate the effect of donor age in canine SCNT efficiency.