231 EXPRESSION PATTERN OF CERTAIN DEVELOPMENTALLY IMPORTANT GENES IN BOVINE NUCLEAR TRANSFER EMBRYOS PRODUCED USING CELL LINES OF DIFFERENT EFFICIENCY

Abstract

Developmental abnormalities associated with the cloning process suggest that reprogramming of donor nuclei into an embryonic state may not be fully completed in most of the cloned animals. One of the areas of interest in this respect is the analysis of gene expression patterns in nuclear transfer embryos to dissect the processes that failed and to develop means to overcome the limitations imposed by these factors. In this study, we investigated the expression patterns of histone deacetylase-1,-2,-3 (HDAC-1,-2,-3), DNA methyltransferase-3A (DNMT3A) and octamer binding protein-4 gene (POU5F1) in donor cells with different cloning efficiencies (low: no-pregnancy, medium: pregnancy but no live birth and high: live birth) and nuclear transfer embryos derived from these cell lines using a real time reverse transcription-polymerase chain reaction (RT-PCR) assay with SYBR green chemistry. Employing standard protocols, we produced nuclear transfer embryos from three different cell lines categorized as having varying efficiencies in supporting development to term. Embryos were collected at morula, blastocyst and hatched blastocyst stages and total RNA was extracted from pools of 4–5 embryos using Absolutely RNA nanoprep kit (Stratagene, La Jolla, CA, USA). Relative level of expression at these stages was analyzed using ΔΔCT method with HH2A as the reference gene and in vitro-fertilized embryos as the control samples. Statistical analysis was performed on ranked expression data employing SAS statistical analysis software procedure ANOVA. Same set of genes were also analyzed on donor cells using standard curve method. All genes investigated were affected by nuclear transfer and followed somewhat altered expression patterns. In general, expression of HDAC genes was elevated especially at the compact morula stage but became comparable to control embryos at the hatched blastocyst stage. DNMT3A expression in NT embryos was lower than in IVF embryos at all stages. POU5F1 transcript levels were also reduced in nuclear transfer embryos at the compact morula and blastocyst stages. The difference, however, disappeared at the hatched blastocyst stage. There was a cell line effect on the expression patterns of all genes investigated. Cell lines efficient in producing offspring tended to resemble control embryos in gene expression patterns compared to inefficient cell lines. These results agree with several studies reporting altered gene expression patterns for certain genes in cloned embryos. Our data also suggest that cell line differences in developmental competency observed in cloning experiments might be related to physiological differences in transcriptional regulation and nuclear remodeling, DNA methylation, and lineage differentiation in embryos cloned from these cell lines.