Aberrant chromatin remodeling has been implicated in the low success rates achieved from cloned embryos. Following fertilization, DNA methylation within a normal embryo is rapidly reduced to a very low level and remains low until the 8–16 cell stage when DNA methylation once again increases. In contrast, the majority of cloned embryos fail to exhibit a similar methylation pattern. This may be due to somatic cell-associated DNMT1s keeping methylation high. However, attempts to chemically modify methylation patterns of donor cells prior to cloning have proven problematic. The objective of this study was to determine if a more natural approach, such as culture conditions, time in culture, and/or cell type, could alter DNMT1 expression in donor fibroblast cells. Two experiments were designed to meet these objectives. Donor fibroblast cell lines were produced from biopsies taken from male and female skin, ovaries, and testes, and were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% fetal bovine serum, 0.1 mM non-essential amino acids, 2 mM L-glutamine, and 0.1 mM β-mercaptoethanol, in a humidified environment of 5% CO2 in air, at 39°C. In Experiment 1, cell lines were maintained at 70% confluence to passage 4, 8, and 12, and analyzed by reverse transcription real-time PCR. In Experiment 2, cell lines were evaluated under 3 culture conditions: proliferating (70% confluence), serum-starved (0.5% FBS), and confluent (100%), and analyzed by reverse transcription real-time PCR. RNA was isolated from cell lines using Trizol reagent (Invitrogen, Carlsbad, CA, USA), reverse transcribed, and analyzed for DNMT1 expression using Taqman real-time PCR, with β-actin as the reference standard. All samples and no template controls were run in triplicate. Final quantitation was done using the comparative CT method, and relative DNMT1 expression was analyzed using one-way ANOVA followed by LS means multiple comparisons. Cell type and passage number had a significant effect on DNMT1 expression. Ovarian fibroblasts had an overall increase in expression in DNMT1 over time (P < 0.05), whereas male skin fibroblasts demonstrated an opposite trend (P = 0.05). Female skin fibroblasts and testes fibroblasts also had a decrease in DNMT1 expression over time, but only approached significance (P < 0.10). For Experiment 2, culture conditions tested did not affect DNMT1 expression for any except one skin cell line. In that case, proliferating cells had significantly higher DNMT1 than quiescent cells (P < 0.005). This research emphasizes the importance of donor cell type and culture effects over time on gene expression. These important aspects should be considered when selecting and growing donor cells to be utilized in somatic cell nuclear transfer.
This project was supported by National Research Initiative Competitive Grant no. 2006-35203-16620 from the USDA Cooperative State Research, Education, and Extension Service and the Florida Agricultural Experiment Station.
Establishing Cell Lines from Fresh or Cryopreserved Tissue from the Great Crested Newt (Triturus cristatus): A Preliminary Protocol
