47 DIFFERENCES IN MITOCHONDRIAL DNA COPY NUMBER AND EPIGENETIC PATTERNS OF MITOCHONDRIA-RELATED GENES IN CLONED COWS FROM THE SAME DONOR CELLS

Abstract

The genetic codes of cloned animals and the donor are identical; however, incomplete reprogramming of donor nuclei during NT causes epigenetic abnormalities in cloned animals. Due to the genetic identity and epigenetic differences among clones, we can study epigenetic effects on the phenotypes by analyzing genetically identical clones. During the NT process, donor cell mitochondria (mt) are transferred into the recipient oocytes and mtDNA heteroplasmy is observed. Previous studies have reported various mtDNA transmission patterns not only in the cloned animal itself but also in the offspring of clones. However, differences in mtDNA copy number in cloned animals have not been reported, especially genetically identical ones. To analyze the genetic effects on mtDNA copy number in cattle, we compared actual mtDNA copy number per diploid genome in various tissues of clones derived from the same donor cells. From 5 genetically identical cloned cows (Japanese Black cattle, ages 68 to 82 months) and 6 non-cloned cows (Japanese Black cattle, ages 52 to 129 months), we isolated DNA from 8 kinds of tissues (heart, lung, liver, kidney, spleen, small intestine, muscle, and spinal cord) and measured mtDNA copy number by using real-time PCR. The absolute copy numbers of 2 mtDNA-encoded genes (COX1 and CytB) and 2 nuclear-encoded genes (H19 and IGF2) were measured and analyzed. To examine the epigenetic effects on mitochondria-related genes, we also analyzed DNA methylation patterns of mitochondria-related gene ANT4 (mitochondrial ADP-ATP translocase) in these tissues by the combined bisulfite restriction analysis (COBRA) method. The actual mtDNA copy number per diploid genome varied in tissues and individuals both in clones and non-clones (average in clones v. non-clones: heart: 11 839 ± 6210 v. 9569 ± 2555; lung: 2027 ± 1153 v. 1383 ± 173; liver: 5644 ± 2278 v. 4799 ± 1848; spleen: 1080 ± 844 v. 393 ± 265; kidney: 7034 ± 4448 v. 2939 ± 784; small intestine: 1330 ± 573 v. 437 ± 171; muscle: 9861 ± 3640 v. 7907 ± 3229; spinal cord: 3961 ± 1819 v. 2756 ± 496). The variability of mtDNA copy number in clones was significantly higher in the lung, spleen, kidney, small intestine, and spinal cord (P = 0.001, 0.026, 0.005, 0.021, and 0.014, respectively; F-test), but not in other tissues. Methylation of the ANT4 gene is quite tissue dependent: hypomethylated in the liver, muscle and spinal cord; moderately methylated in the heart, lung, and kidney; and highly methylated in the spleen and small intestine. The methylation patters of ANT4 were not different between clones and non-clones. These results suggest that mtDNA copy number is more influenced by nongenetic factors than genetic background.