Practical application of animal cloning by somatic cell nuclear transfer (SCNT) has been hampered by extremely low success rate. Most clones die before birth and survivors frequently display abnormalities. It is speculated that epigenetic reprogramming is somehow defective in reconstituted embryos (Reik W et al., 2003 Theriogenology 59 21–32; Han YM et al., 2003 Theriogenology 59, 33–44). It is likely that placental anomalies are directly or indirectly responsible for the death of cloned fetus and neonates. To address this question, we analyzed protein patterns of two placentae obtained after postnatal death of fetuses from SCNT of Korean Native Cattle and two normal placentae obtained after birth of AI fetuses. Global proteomics approach was employed by using 2-D gel electrophoresis and mass spectrometry to separate the different placenta proteins. Proteins within an isoelectric point range of 4.0 to 7.0 and a molecular weight range of 20–100kDa were analyzed by means of 2-D gel electrophoresis with three replications of each sample. The stained gels were scanned and calibrated at an optical resolution of 63.5μm/pixel using a GS-710 (Bio-Rad Laboratories, Hercules, CA, USA). Approximately 480 spots were detected in placental 2-D gel stained with coomassie-blue. Then, image analysis by Malanie III (Swiss Institute for Bioinformatics, Geneva, Switzerland) was performed to detect variations in protein spots between normal and SCNT placentae. In the comparison of normal and SCNT samples, at least 15 protein spots were identified as regulated differentially. Using MALDI-TOF-MS (PerSeptive Biosystems, Framinham, MA, USA), 10 spots were identified as up-regulated proteins in SCNT placentae including BPLP-I, Rho GDI 2, osteoclast stimulating factors, SM22, 60S Acidic Ribosomal and Protein P2, whereas five spots were down-regulated proteins such as Peroxiredoxin 2. Mass spectrometry with sequencing was used to further analyze the uncharacterized proteins. Most identified proteins in this analysis appeared to be related to cell proliferation and differentiation, fetal growth and development or metabolism. Further, specific functions of proteins in placenta have been investigated at the molecular levels during pregnancy.
Animal cloning experiments still banned in Italy