152 PROLIFERATION- AND SENESCENCE-RELATED GENE EXPRESSION IN CANINE FIBROBLASTS DERIVED FROM DOGS OF DIFFERENT AGE

Abstract

In pig, cattle and mouse cloning, the age of the cell donor can influence cloning efficiency. However, little is known regarding the effects of the cell donor’s age related to cell proliferation and senescence and its importance for canine cloning. Therefore, the aim of this study was to investigate the effect of cell donor age on growth pattern and doubling time of cell populations, cell size, viability, and senescence-related gene expression (p53, p16, p21, Rb1, and E2F1) in canine fibroblasts. Cells were derived from 1-year-old and 7-year-old dogs. To obtain the fibroblasts, primary culture was performed using ear biopsies from three 1-year-old dogs (1yr) and three 7-year-old dogs (7yr), and cells at passage 2 to 6 were used for somatic cell cloning. Cells were plated at 1 × 10⁵ cells well^–1 and harvested every 24 h for 6 days. Cell number was determined for measurement of growth pattern and doubling time. The harvested cells were stained with trypan blue and their size and viability were analysed using a Countess automated cell counter (ThermoFisher Scientific, Waltham, MA, USA). Gene expression was measured by RT-qPCR. Cell proliferation and gene expression experiments were technically replicated 3 and 4 times, respectively. Statistical analysis was performed using GraphPad Prism (GraphPad Software, San Diego, CA, USA) and t-test (P < 0.05) was used. Growth curves in both groups showed typical S-shape and at Day 6, 1yr samples had significantly higher cell numbers compared with 7yr samples. For doubling times, passages 2 to 5 of 1yr (29.8 ± 4.4 h, 41.0 ± 1.6 h, 49.5 ± 4.8 h, and 71.3 ± 8.4 h) were significantly shorter than those of 7yr (47.0 ± 4.5 h, 57.1 ± 2.3 h, 65.8 ± 1.8 h, and 126.4 ± 12.4 h). Cells from passage 6 of 7yr and passage 7 of 1yr did not reach confluence at Day 6. Cell size of 1yr (14.1 ± 0.2 µm) was significantly smaller than that of 7yr (15.3 ± 0.2 µm). Additionally, there was no significant difference in cell viability between 1yr (90.7 ± 0.8%) and 7yr (88.7 ± 0.7%). However, mRNA expression of p53, p21, and Rb1 was significantly lower in 1yr cells compared with 7yr cells. The expression level of E2F1 was significantly higher in 1yr cells. There was no significant difference between 1yr and 7yr cells for p16 expression. In conclusion, proliferation analysis indicated that somatic cells derived from old donor dogs reach a state of senescence more rapidly compared with those of young donor dogs. In addition, analysis of gene expression demonstrated that cells from old donor dogs have significantly increased p53, p21, and Rb1 expression levels, which indicate senescence. Further studies are needed to produce dogs using 1yr and 7yr cells to evaluate the effect of donor aging in canine somatic cell nuclear transfer efficiency.

This study was supported by RAD (#PJ0109282015), IPET (#311062-04-3-SB010), Nature Cell, Research Institute for Veterinary Science, (Natural Balance Korea or Nestle Purina Korea) and the BK21 plus program. We thank Barry D. Bavister for his valuable editing of the manuscript.