SOMATIC CELL NUCLEAR TRANSFER USING DONOR CELLS SELECTED FOR DNA METHYLTRANSFERASE 1 (DNMT1) EXPRESSION

Abstract

Bovine nuclear transfer (NT) embryos and fetuses often display abnormalities that are not observed in their in vivo produced counterparts, and are attributed to large offspring syndrome. Gene expression is altered in NT embryos and is thought to be due to inadequate reprogramming of the somatic cell nucleus. It is known that the embryonic genome must be rapidly demethylated, but often does not occur normally in NT embryos. This may be due to the introduction of Dnmt1s into the NT embryo, the enzyme responsible for maintaining DNA methylation (DM) in somatic cells, but which is never found in early embryos. Chemicals, such as 5azaC, have been used to artificially reduce DM, but have proven problematic in NT. We previously showed that culture without chemicals can be used, with cell type and time in culture affecting expression of Dnmt1 in donor cells. Here we tested the effects of using donor cells selected for Dnmt1 expression (low vs. high) as a means to reduce DM in NT embryos. Cell lines were grown to passage 4, 8 and 12, diluted out and frozen in individual use aliquots for later use. An aliquot of each cell line was taken for RNA isolation, and Dnmt1 expression analysis using Real-time RTPCR. Two cell lines with the same genotype, but at different passages were selected as having higher (HD) and lower Dnmt1 (LD) expression for use in NT experiments. Cumulus oocyte complexes were retrieved by slashing 2–8mm follicles on ovaries obtained from the abattoir, and were matured for 17–19h. Metaphase II oocytes (n=874) were selected for polar body extrusion and were enucleated for use in NT. Cells were transferred from either HD or LD groups, and fused. Fused couplets were activated with ionomycin and DMAP using standard protocols, and cultured within treatment in KSOM to d7 (activation=0). Global DM was evaluated in NT (LD and HD) and in vitro produced (IVP) control embryos (2-cell, 8-cell and blastocyst stages, 10/treatment and stage) by immunocytochemistry using 5MeC antibody (Calbiochem) and a FITC-conjugated secondary antibody (Santa Cruz). IgG and PBS served as negative controls to show antibody specificity. Pixel data were collected using AxioVision software (Zeiss) and were analyzed by LSMeans ANOVA using SAS, and significance was deemed p<0.05. There was significantly less DM in 2-cell IVP when compared to either LD or HD NT embryos (p<0.05). By the 8-cell stage, NT produced with LD had lower DM than 8- cell HD NT (p<0.05), but was still higher than 8-cell IVP (p<0.05). NT blastocysts produced with LD had DM similar to IVP (p>0.05), and both were lower than HD NT blastocysts (p<0.05). These data indicate that cell lines selected for reduced Dnmt1 expression can reduce global DM in the developing NT embryo. However, DM was not reduced to the levels found in IVP embryos at either the 2- or the 8-cell stages. Future efforts will need to determine if these reductions are adequate and allow for normal development in vivo, or if additional efforts will need to be made to further mimic normal embryonic reprogramming events. This project was supported by National Research Initiative Competitive Grant no. 2006- 35203-16620 from the USDA Cooperative State Research, Education, and Extension Service. (platform) (platform)