260 COMPREHENSIVE IDENTIFICATION OF RELATIVE GENES CAUSING ABNORMAL DEVELOPMENT OF BOVINE EMBRYOS PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER

Developmental failure of a cloned animal using somatic cell nuclear transfer (SCNT) procedures is considered to be the result of abnormal expression of developmentally important genes caused by incomplete reprogramming of the donor cell nuclei. However, there are few reports about stage-specific gene expression during cleavage progression of cloned embryos. The aim of this study was to identify using fluorescein differential display method, the differentially expressed genes in cloned embryos at early developmental stages compared with those produced by in vitro fertilization. Bovine cumulus-oocytes complexes (COCs) were aspirated from follicles (2-8 mm in diameter) of slaughterhouse ovaries and cultured in TCM-199 supplemented with 10% fetal calf serum (FCS) for 18 h for somatic cell nuclear transfer (NT) or 24 h for in vitro fertilization (IVF) at 39�C. Removal of oocyte nuclei for NT was performed by squeezing out a small amount of the cytoplasm laying beneath the first polar body by means of a glass needle. Donor cells for NT were obtained from skin cells of an adult cow and cultured in DMEM supplemented with 10% FCS. After the transfer of somatic cell into enucleated oocytes, DC electric pulses at 200 V/mm for 2 � 10 �s were used for fusion, and the reconstructed embryos were treated with 10 �g/mL cycloheximide for 6 h. The embryos were then cultured for 120 h (morula stage) or 168 h (blastocyst stage) in modified SOF medium under 5% CO2, 5% O2 and 90% N2 at 39�C. Total RNA obtained from NT and IVF embryos were analyzed by differential display RT-PCR (DDRT-PCR) as previously described (Minami et al. 2001 Biol. Reprod. 64, 30-35). We obtained several differences in gene expression patterns between NT and IVF embryos at the morula and blastocyst stage. A total of 52 cDNA fragments were isolated and analyzed. Semiquantitative analysis revealed that some genes (NADH dehydrogenase subunit 1, SR rich protein, KIAA0107, ribosomal protein L19) were highly expressed in IVF embryos compared with NT embryos, whereas other genes (CASK) were highly expressed in NT embryos compared with IVF embryos. These results indicate that the differentially expressed genes observed in NT embryos may be representative of marker genes for the production of normal NT offspring and DDRT-PCR procedure is quite useful for identification of several genes that are differentially expressed between NT and IVF embryos.Although the detailed function of the genes and their products remains to be determined, it is likely that the reprogramming mechanisms can be elucidated genetically by the analysis of differentially expressed genes in the future.