RESULTS

All deceased cloned cattle died within 48 h of birth, and all had aberrations in many of their organs. The principal abnormalities at necropsy are shown in Table 2. Seven hearts from deceased clones were abnormal, displaying compensatory cardiac hypertrophy, vascular incompetence and patent foramen ovale, cardiac hemorrhage, and necrosis. At birth, atelectasis was observed in the lungs of five cloned calves. Other common abnormal characteristics included thickening of the lung alveolar wall, pulmonary hemorrhage, inflammation, and congestion. Hepatomegaly and congestion were found primarily in livers of cloned calves. Oddly, AF5 had six lung lobes, which did not connect to each other (Fig. IE), and the kidney of FF2 had much fat, both inside and outside (Fig. 1C). AF4 had only a small amount of brain tissue, with necrosis, hemorrhage, and edema (Fig. 1A). The heart of AF3 was enlarged (0.4 kg) and had a patent foramen ovale (Fig. IB). A large cyst was found on the liver of AF1 (Fig. IF). Figure ID shows the heart of AF1, with cardiac muscle putrescence and congestion. These findings suggest serious aberrations in the organs of cloned bovines that die soon after birth.

Amplification products were identified by melting curve profile analysis and confirmed by gel electrophoresis and by sequencing. Figure 2 shows photographs of representative gels from real-time RT-PCR analysis of 420 base pairs (bp) of Gapdh, 398 bp of (i-actin, 226 bp of Xist, 340 bp of VEGF, 425 bp of Hsp70.1, 239 bp of FGF10, 220 bp of PCAF, 220 bp of FGFR2, 249 bp of PDGFRa, and 223 bp of BMP4. The final relative abundances of each target gene normalized by Gapdh were the same as the results normalized by f-actin.

Gene expression in each of the two types of clones was first compared with that of controls. The relative transcription abundances of PCAF, Xist, FGFR2, PDGFRa, FGF10, BMP4, Hsp70.1, and VEGF in liver, heart, lung, spleen, kidney, and brain of cloned calves from both types of donor cells as compared to controls are shown in Table 3. Aberrant expressions of genes were found in all studied tissues. The majority of aberrantly expressed genes were common to both types of clones, whereas some were dysregulated either in AF-derived or in FF-derived clones. In heart, five abnormally regulated genes were found, in which VEGF (P < 0.05), BMP4 (P < 0.01), and PCAF (P < 0.05) were elevated in both types of clones compared to controls, whereas FGF10 showed a higher level (P < 0.05) only in FF-derived clones and Xist a higher level (P < 0.01) only in AF-derived clones. In brain, a higher level of VEGF (P < 0.01) and a lower level of Hsp70.1 (P < 0.01) were observed in both types of clones, but BMP4 was elevated (P < 0.01) only in AF-derived clones. In kidney, a lower expression level of PDGFRa (P < 0.01) was seen in both types of clones, but FGF10 showed a lower level (P < 0.05) only in FF-derived clones. In spleen, BMP4 was reduced noticeably (P < 0.01), and VEGF was elevated (P < 0.05), in the two types of clones; however, PCAF fell (P < 0.05) only in AF-derived clones. Three genes had aberrant expression in lung: Hsp70.1 (P < 0.01) and BMP4 (P < 0.05) were elevated in both types of clones, and PDGFRa was reduced (P < 0.05) in FF-derived clones. For liver, PCAF (P < 0.05) was elevated in both types of clones, and Hsp70.1 showed a higher level (P < 0.01) only in AF-derived clones.

In addition to comparing the gene expression of two types of clones with those of controls, we compared the gene expression between the two types of clones with each other. The expression of only four genes showed a significant difference between AF-derived and FF-derived clones. The expressions of BMP4 in brain, PDGFRa in lung, and Xist in heart showed a higher level (P < 0.01) in AF-derived clones than in FF-derived clones. Conversely, FGF10 in heart showed a lower level (P < 0.05) in AF-derived clones than in FF-derived clones.

Although the difference in the expression of some genes between the clones and the normal controls did not reach statistical significance in some organs, the variability among the individual clones was noticeable. The expression of Hsp70.1 showed noticeable variation in heart, liver, spleen, and kidney in the clones (Fig. 3A). For example, the expression of Hsp70.1 in the lung of FF3 and the kidney of FF1 was approximately sevenfold higher than in controls, but the expression of Hsp70.1 was not detected in the heart of either FF1 or FF2, in the spleen of either AF4 or AF5, or in the liver of either FF2 or FF3. In the case of FGF10, wide variations were also observed in most studied tissues from the dead cloned calves (Fig. 3B). In addition to no expression of FGF10 in the spleen and liver of FF2, very low expression levels were observed in the lung of both FF2 (0.003) and FF3 (0.006) and in the spleen of both FF1 (0.09) and AF3 (0.11) compared to the controls; however, a high level was observed in the heart of FF2 (6.61-fold), the liver of AF5 (5.73-fold), and the brain of AF5 (5.37-fold). Expression of BMP4 in the spleen of FF3 and of PDGFRa in the heart of AF3 was absent. The expression variations of Xist and FGFR2 in deceased cloned animals were smaller in eight candidate genes.

Results of all the dysregulated genes, according to the organ studied, are summarized in Table 4. For the studied genes, kidney was the organ that was least affected (two genes) by gene dysregulation, whereas heart was the organ that was most affected (five genes). Most cases of dysreg-ulation (12 of 19) were up-regulation, but PDGFRa only showed down-regulation. VEGF, BMP-4, PCAF, and HSP were extremely dysregulated, whereas the other four genes had a low level of gene dysregulation.

Table 1.
table1Aberrant Gene Expression in-1
The primer pair to detect these mRNAs was first designed from the given human and sheep separate heterologous sequence. The product was sequenced, and the resulting bovine-specific sequence was used to create the primer pair employed to detect the transcript of interest.

Table 2.
table2Aberrant Gene Expression in-2

Fig1Aberrant Gene Expression in-3
FIG. 1. Pictures of representative abnormalities found in cloned calves that died soon after birth. A) Brain of AF4, with only a small amount of brain tissue and with necrosis, hemorrhage, and edema. B) Heart of AF3, of an enlarged size (0.4 kg) and with a patent foramen ovale. C) Kidney of FF2, with fat inside and outside. D) Heart of AF1, with cardiac muscle putrescence and congestion. E) Lung of AF5, with six lung lobes that do not connect and with congestion. F) Liver of AFI, with a large cyst.

Fih2Aberrant Gene Expression in-4
FIG. 2. Photographs of representative gels from real-time RT-PCR analysis. Lane 1: 398 bp of fi-actin; lane 2: 223 bp of BMP4; lane 3: 249 bp of PDGFRa; lane 4: 220 bp of PCAF; lane 5: 240 bp of VEGF; lane 6: 220 bp of FGFR2; lane 7: 425 bp of Hsp70.1; lane 8: 226 bp of Xist, lane 9: 420 bp of Gapdh; lane 10: 239 bp of FGF10.

table 3.
table3Aberrant Gene Expression in-5
a The relative transcript levels of genes in deceased calves derived from AF and FF compared with normal controls (n = 3; the relative transcript levels of normal control were normalized to 1). Values are presented as the mean ± SEM. b P < 0.01. c P < 0.05.

Fig3Aberrant Gene Expression in-6
FIG. 3. The relative transcript levels of Hsp70.1 (A; in heart, liver, spleen, and kidney) and FGF10 (B; in heart, liver, spleen, lung, kidney, and brain) of deceased calves derived from two kinds of donor cells—AF (n = 5) and FF (n = 4)—compared with controls (n = 3, normalized to 1), which are represented by the dashed line.

Table 4.
table4Aberrant Gene Expression in-7
Up, Up-regulation in both clones derived from AF and from FF cells; Down, down-regulation in both clones derived from AF and from FF cells; Up (AF), up-regulation in clones derived from AF cells; Up (FF), up-regulation in clones derived from FF cells; Down (AF), down-regulation in clones derived from AF cells; Down (FF), down-regulation in clones derived from FF cells. b Total.