Effect of cell culture duration (passage numbers)
Our group was the first to directly compare passage effect of donor cells on the outcome of nuclear transfer [16]. In our study, we found that cells of later passages (up to 15) could also support clone development to full term (Table 5).
Table 5  Cloning efficiency of cells at different passages
No. Passage  No. NT  No. (%) fused  No (%) cleaved  No. (%) blastocyst
5  288  114 (40)  75 (66)  24 (21)a
10  269  115 (43)  72 (63)  43 (37)b
15  264  109 (41)  81 (74)  36 (33)b
Numbers with different superscripts within columns are significantly different (P < 0.05). Comparable to our findings were those of Arat et al. [40] who established a primary cell line from granulosa cells and transfected them with the green fluorescence protein (GFP) gene. Non-transfected cells were used for cloning between passage 10 and 15 as either serum-starved or serum-fed donor cells. There were no differences in development to the blastocyst stage for nuclear transfer embryos from transfected or non-transfected or from serum-starved or serum-fed cells. Blastocyst development rates of embryos produced from donor cells at passage 15, however, were significantly higher than those produced with cells at passage 10, 11, and 13. Developmental competence of later passages, up to 16 [54] and as high as 36, from fibroblast from a cloned fetus [41], have also been reported.
The demonstration that later passages can support clone development is essential for utilizing somatic cloning for gene-knockout studies, in which single cells must be clonally expanded to generate sufficient cells for nuclear transfer [7]. These afore-mentioned studies suggest that cells of higher passages were receptive to nuclear reprogramming. Additional support for this hypothesis comes from a recent study by Enright et al. [59] who showed that cells of later passages contain less epigenetic modifications, i.e., their histones are more acetylated than in earlier passages. This observation agrees with an earlier notion that in vitro culture of cells can induce expression of genes that were not expressed before culture [60,61]. Furthermore, Hills et al. [62] reported that a greater proportion of late passage cells (passage 18), vs. earlier passage cells (passage 2), were found to be in G0/G1 whether or not they were in serum-starved culture conditions.