Analysis of the Telomere Shortening in the Cloned Caprine Cultured Cell Line, CPF-1, Derived from Placenta of Shiba Goat. (Capra hircus).

Abstract

As a prerequisite for the production of cloned goats by nuclear transfer from the cultured somatic cells, we established cloned fibroblast cell lines, CPF-1 and other related CPF subclones from the placenta of the Shiba goat. CPF-1 cells ceased to proliferate at and about the 51st population doubling level (PDL) when the morphological signs of senescence, such as an unusual increase in cell volume, became obvious. The mean surface area of a cell at 51PDL was about 3 times greater than that of an early-passage cell (3PDL) (p<0.001). When the CPF-1 cells at 14PDL were subjected to chromosomal analysis, 49.3% of the cells turned out to be of normal diploid chromosomal constitution (2n=60). All the chromosomes were of acrocentric types. Part of the reason for the rather high incidence of aneuploidy might be due to the well-known technical difficulty in preparing good chromosomal spreads in the caprine cells. We analyzed the rates of telomere length shortening in the CPF-1 cells, according to the number of passages and culture periods. The telomere length in the late-passage cells became approximately 1/2.5 of that of the early-passage cells (p<0.05). The estimated mean rate of telomere shortening in the CPF-1 cells was approximately 260 bases per PDL and faster than the values (50-200 bases/PDL) so far reported in the literature for other types of cells. Furthermore, in the CPF-1 cells the replicative capacity was exhausted when the mean telomere length reached approximately 7.5 kb which is significantly longer than the values generally obtained in other cultured cell lines. The reasons for the observations have not yet been clarified.