Gene Expression in Cloned Bovine Fetal Liver

Abstract

Nuclear transfer (NT) is a method of animal reproduction that bypasses fertilization and propagates known combinations of genes. Currently NT is an inefficient process. Attempts have been made to increase the efficiency of this procedure, but most have been deemed unsuccessful. Some problems associated with NT are unusually large birth weights, and physical abnormalities in developing liver, heart, and brain. Despite numerous studies performed on NT animals, the factors behind the anomalies remain unknown. It is possible that nuclear reprogramming is the basis of poor development rates, meaning, when the donor cells are fused with enucleated eggs the nuclei may not regain the full ability to direct cell differentiation in subsequent mitotic divisions. If reprogramming is not carried out precisely, then some genes may not be correctly expressed in NT animals. The purpose of this study was to determine if differential gene expression between the livers of NT fetuses when compared to an embryo transfer (ET) derived fetus could be detected and the genes identified. An Angus fetus at 45 d of gestation was collected and a non-clonal cell line established for use as NT donor cells. Two NT fetuses were propagated and compared to the original. Differential Display Reverse Transcription Polymerase Chain Reaction (ddRT-PCR) was used to identify genes that were differentially expressed. Differentially abundant cDNAs were subcloned, sequenced and their corresponding mRNAs were verified by semi-quantitative RT-PCR. Twenty-three Expressed Sequence Tags (ESTs) were sequenced in Bos taurus and submitted to GenBank. The results of ddRT-PCR identified 39 genes/ESTs that were potentially differentially expressed. Fifteen of the genes were tested by semi-quantitative RT-PCR, but no significant differences were detected.