49 TRANSCRIPT LEVEL OF mRNA IN BOVINE CLONED OR RE-CLONED EMBRYOS

Abstract

Gene expression in embryos played important roles during preimplantation development and had the potential to be used as an indicator for embryo viability. The purpose of this study was (1) to compare the developmental competence of cloned, or re-cloned embryos (Experiment 1); (2) to analyze the transcripts level of the related implantation, metabolic, and imprinting genes in IVF, cloned and re-cloned embryos (Experiment 2). The SCNT was performed according to the established system in our laboratory (Theriogenology 2006 65, 1800–1812). For producing cloned embryos, fetal fibroblasts as donor cells were used and a viable cloned calf was born. Recloned embryos derived from ear fibroblasts of the cloned calf, genetically same with donor fetal donor cells, were produced. The couplets were fused, chemically activated, and cultured in modified synthetic oviduct fluid (mSOF) for up to 7 days. The developmental competence up to blastocysts was observed under a microscope. The implantation (Bax, E-cad, If-tau, Hsp 70, Igf2r, and DNMT1), metabolic [LDHA (Lactate Dehydrogenase A), G6PD (Glucose-6 Phosphate Dehydrogenase), PGK (Phosphogycerate Kinase), Na/K ATPase, and Glut-1], and imprinting [GNAS (guanine nucleotide binding protein, alpha stimulating), UBE3a (ubiquitin protein ligase E3A), Mest (mesoderm specific transcript), SNRPN (small nuclear ribonucleoprotein polypeptide N), and Ndn (necdin)] genes were selected. The relative abundance (ratio to GAPDH mRNA) of gene transcripts in blastocysts was measured by conventional semi-quantitive RT-PCR. In Experiment 1, development competence of SCNT pre-implantation embryo was not different between cloned or re-cloned embryos (26% vs. 22%). In Experiment 2, the relative expression of Bax, Hsp70, If-tau, and Igf2r transcript was not different in IVF, cloned, and re-cloned embryos. Expression of E-cad and DNMT1 was higher in re-cloned embryos than any other group. Transcripts levels of LDHA, Na/K ATPase, and Glut-1 showed the similar relative abundance in IVF, cloned and re-cloned embryos. Expression of G6PD and PGF was increased in re-cloned and cloned embryos, respectively. Compared to IVF and cloned embryos, in re-cloned embryos, relative abundance of related imprinting genes (Ube3a, Mest, SNRPN, and Ndn) was increased. In conclusion, this study demonstrated that, whereas developmental competence of cloned or re-cloned embryos was not different, gene transcript levels were observed differently. It was suggested that alteration of gene expression in the re-cloned embryos derived from the cloned calf, genetically same with initial donor cells, might have affected the fetal development and births of re-cloned offspring.