The efficiency of animal production using somatic cell NT is very low. The doner nucleus must establish normal expression patterns to ensure that the reconstructed embryo develops successfully. To determine the possible genetic causes of death in animal production using NT techniques, the transcription of eight genes (Xist, VEGF, Hsp70.1, FGF10, PCAF, FGFR2, PDGFRa, and BMP4) was compared in the major internal organs of deceased clones and normally reproduced control calves using real-time RTPCR. These genes are known to have important functions during development of the embryo and organogenesis in mammals.

In female mammals, dosage compensation is gained by X-chromosome inactivation, with random transcriptional silencing of one of the two X chromosomes in female cells during late blastocyte development, and the Xist, (X-inac-tive-specific transcript) plays a crucial role for XCI. Previously, patterns of XCI have been reported to be normal in cloned mice, but in the present study, expressions of Xist in the heart of AF-derived clones was higher than in the controls. In addition, overexpressions of Xist were found in cloned bovine embryos, and hypome-thylation of Xist was observed in deceased cloned bovines. Overexpression of Xist in the hearts of clones that died soon after birth may result in aberrant reactivation of the silent X chromosome, where incomplete nuclear reprogramming may generate abnormal epigenetic marks on the X chromosomes and then affect the normal patterns of XCI.

In the present study, aberrant expression of Hsp70.1 was observed in the lung, brain, and liver of cloned animals. Heat shock proteins are stress proteins, and their binding of apoptosis-inducing factor (AIF) inhibits the nuclear import of AIF and, therefore, protects the embryo from undergoing apoptosis. The transcripts of Hsp70.1 were not detected in bovine blastocysts from NT. The present results suggest that the NT embryos did not copy in the adverse environment. Consequently, the normal expression of Hsp70.1 was affected, disrupting the viability of offspring from the NT.

Both DNA methylation and histone acetylation on nuclear chromatin play important roles in the regulation of gene activation, where histone acetylation is involved in the inheritance of cell memory. Several reports have shown inefficient demethylation and inappropriate re-establishment of DNA methylation during somatic NT PCAF is a transcriptional coactivator with intrinsic histone acetylase activity. It contributes to transcriptional activation by modifying chromatin and transcriptional factors in addition to its role in transcriptional activation, cell-cycle arrest, and cell differentiation in cultured cells. In the present study, aberrant expression of PCAF was observed in some organs of cloned bovines, which agrees with the research of Enright et al., who concluded that histone acetyla-tion status is remodeled in cloned bovine embryos. They also found that histone deacetylase inhibitor and DNA methyl-transferase inhibitor can improve the reprogramming ability of donor cells. Therefore, NT leads to epigenetic abnormalities that affect the normal expression of PCAF.

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, has been implicated in the regulation of blood vessel formation (i.e., vasculogenesis and angiogenesis). Development of the cardiovascular system depends on the generation of precise VEGF concentration gradients. A decrease in the amount of VEGF produced during development of the embryo may lead to decreased angiogenesis, with fatal consequences. VEGF modulates early heart valve formation. Significantly higher expression of VEGF was seen in cloned animal heart, spleen, and brain compared to controls. Coincidentally aberrant valvulogenesis was seen in cloned heart, and congestion and hemorrhage were seen in some cloned heart, brain, and spleen. The aberrant expression of VEGF may contribute to these defects as reported in the clones that died soon after birth.

In the cloned animals, a significantly lower expression of PDGFRa was observed in the kidney and lung compared to controls. PDGFRa plays an important role in lung development and has a relationship with renal development and disease. The results suggest that NT leads to aberrant epigenetic activity affecting the normal expression of PDGFRa, particularly in the lung and kidney of cloned bovines.

BMPs, a subgroup of the transforming growth factor-p superfamily, have been found to play roles in many embryonic patterning events and in all aspects of embryonic development and organogenesis. In the present study, the expression of BMP4 showed a higher level (P < 0.01) in cloned lungs compared to control calves. Aberrant morphogenesis was found in the lungs of seven clones. Both AF5 and FF2 showed very poor lung mor-phosis: AF5 had six lung lobes, which did not connect to each other, and FF2 had only one lung lobe. Previous researchers showed that BMP4 was one of the key growth factors essential for lung development and that overexpression of BMP4 caused abnormal lung morphogenesis, with cystic terminal sacs and inhibition of epithelial proliferation. Therefore, aberrant expression of BMP4 may contribute to lung abnormalities in cloned animals. Proper septation and valvulogenesis during cardiogenesis depend on interactions between the myocardium and the endocardium. BMP4, as a signal from the myocardium, directly mediates atrioventricular septation. The endocardial cushion, cardiac valve, and semilunar valve maturation are controlled by BMP-signaling pathways. The normal expression of BMP4 plays a major role in the atrioventricular septation of the mouse heart. In the present research, the expression of BMP4 showed a higher level (P < 0.01) in cloned hearts with valvular incompetence, patent foramen ovale, or enlarged right ventricle, which suggests that aberrant expression of BMP4 may also contribute to the heart abnormalities of cloned animals.

In cloned bovines, age of the fibroblast donor cell has been reported to have no effect on the in vitro development of bovine NT embryos, but other reports indicate that clones derived from adult cells frequently abort during the later stages of pregnancy and that calves developing to term show a higher number of abnormalities than those derived from newborn or fetal cells. The present results suggest that the age of different donor cells affected the gene expression in cloned bovines, which is consistent with the results of NT experiments in the mouse. Many other genes are involved in mammalian embryonic development and organogenesis, and further research concerning these genes will increase our understanding of nuclear reprogramming events following somatic cell NT, embryonic development, and organogenesis.