The initial expectation for cloned animals was that a group of animals with identical genetic constitution (clones) would have reduced variability associated with all traits when compared with offspring from naturally bred females. The aim of these experiments was to determine to what extent healthy adult clones are affected by the cloning process and whether all phenotypes or traits are affected equally. This information is critical for those proposing to use clones to reduce variability in experimental animals and for those attempting to generate cloned pets with identical phenotypes. Overall, results of the present study indicated a high degree of variation among cloned pigs in some of the specific traits examined. Our experimental design is unique in that it allowed comparison of the degree of variation between clones and controls; we used age-, breed-, and sex-matched clones and control animals housed under identical conditions. Such an experimental design allows the differentiation between environmental effects and effects due to the cloning process itself.
To ensure that the results obtained were not affected by mitochondrial background, we sequenced the D-loop regions of both cloned and control pigs. As previously described [16
], oocytes used to generate these clones were purchased from a commercial supplier that collects oocytes from slaughterhouse material. Thus, oocyte populations represent multiple maternal sources and potentially different mitochondrial types. To separate any potential mitochondrial effects from the epigenetic effects due to methylation differences, the polymorphic hypervariable D-loop mitochondrial region of clones and controls was sequenced. The D-loop region is the site of replication, has a mutation frequency 1000-fold greater than that of the rest of the mitochondrial genome [17
], and therefore is a good indicator of the variability in the remainder of the mitochondrial genome. Little variability among animals was detected, with some cloned and control pigs having identical mitochondrial genotypes in the D-loop regions. This finding indicates that the mitochondrial backgrounds for cloned and control pigs were very similar and unlikely to result in major phenotypic differences. Conservation of mitochondrial background is not surprising, based on the high degree of selection within maternal lines in commercial swine herds and the low degree of mitochondrial variation in swine breeds in general [18
Examination of body weight () indicated that cloning did not reduce the degree of variation associated with this trait, and, with the exception of one cloned pig, values for cloned and control pigs overlapped and were in the normal range for swine at that age. Although the environment can have a significant effect on the weight of an animal, both control and cloned pigs were maintained in the same environment, with the expectation of greater variability in control than in cloned pigs because of higher genetic variation.
When the phenotypic analysis was extended to blood parameters, two overall classes of traits were observed. In one class, there was reduced variation in cloned pigs, and in the other class there was as much variation in cloned as in control pigs. Because all pigs were maintained in the same environment, these results suggest that those traits showing reduced variability in cloned pigs are under tight genetic control. In some cases, the variability within traits for cloned pigs, as determined by relative CVs, was less than half that for control pigs (). This finding is remarkable considering the small number of animals available for this study. Because all pigs were subjected to the same environment, it is unlikely that this reduction in variability is due to an environmental factor; it is more likely due to reduced genetic variability in the clones. Other phenotypes, however, were highly variable among the cloned pigs, suggesting that these traits are more susceptible to aberrations introduced during the cloning process or to an overriding effect of some environmental factor on genetic variability of the clones (either the microenvironment or the initial uterine environment).
Results for two related parameters, T3 and cortisol, were unusual. At 15 wk, variation was similar in cloned and control pigs, but at 27 wk variation was lower cloned pigs, although mean values were not significantly different. Thus, there was no difference between the controls and clones in overall T3 and cortisol concentrations at 27 wk (); however, cloned pigs showed a reduction in the variability of these hormone concentrations. This reduction may be due to an adaptation of the cloned pigs to the environment or to the sampling process, which was more homogenous than that for the controls, suggesting that adaptability to stress responses has a strong genetic component. However, as for all other phenotypic traits examined, both cloned and control pigs were subjected to the same environmental stimuli and were balanced with respect to sex, breed, and age.
Comparison of phenotypes based on blood parameters and body weights indicated two classes of traits: those not affected by cloning and having decreased variability in cloned compared with control pigs and those in which there was equal variability in cloned and control pigs. One interpretation of these results is that some traits, under tight genetic control and not heavily influenced by epigenetics, show a drastic reduction in variability as expected for genetically identical animals. Other traits, however, either because of epigenetic disregulation introduced during the cloning process or susceptibility to very strong environmental effects not accounted for, exhibit a high degree of variability regardless of genotypic similarities. These findings indicate that cloned animals can indeed be utilized when researchers want to reduce the size of an experimental group of animals, but only if the phenotype being examined has reduced variability in clones. For other phenotypes, cloned animals will have no advantage over controls.
In addition to the blood profiles, clonal differences were observed in skin type, hair growth pattern, and number of teats, supporting the observation that cloning creates variation that is independent of genetic background. For number of teats, a recent whole-genome scan for quantitative trait loci (QTL) in swine revealed that three loci, two of which were imprinted, significantly affect this trait [17
]. Because cloning is known to affect imprinted genes [3
], the differences in number of teats in cloned pigs could be due to aberrant expression of the imprinted QTL loci associated with this trait.
These additional trait differences reinforce our previous observations that the cloning procedure can affect traits in some but not all clones. Although it is difficult to differentiate between epigenetic disregulation and environmental effects on blood parameters, it is unlikely that environmental effects that cannot be controlled would have modified traits such as number of teats, hair growth patterns, and skin types.
To determine whether epigenetic disregulation, as represented by CpG methylation, was involved in phenotypic variability in cloned pigs, the methylation status of two repeats, one located in euchromatic regions (PRE-1 SINE) and one located in heretochromatic regions (centromeric satellite), was determined. We detected a differences in methylation status for one of the CpGs in the euchromatic regions and an increase in variation in degree of methylation in cloned pigs. Although it is not possible to prove cause and effect between hypermethylation of a region of the PRE-1 SINE marker and variation in any of the phenotypes measured, the results indicate that the cloning process creates both an abnormal methylation pattern, as determined by hypermethylation of CpG 13 in PRE-1 SINE, and a random variable methylation pattern in several regions, as determined by increased variability in percent methylation in several CpG groups of PRE-1 SINE and one CpG in the centromeric satellite region.
The methylation differences between cloned animals and offspring from naturally bred animals contrast with differences previously described [11
], in that the differences we observed were relatively small. Previous epigenetic analyses have been carried out in animals or tissues with obvious developmental defects, whereas we compared cloned pigs with no apparent developmental defects. Thus, the epigenetic disregulation in normal ‘‘healthy’’ clones should be lower than that in clones that die in utero or are born with severe defects. In short, our results reinforce the hierarchical effects of the nuclear transfer process, with both generalized effects, as reflected by the hypermethylation of CpG 13 of the PRE-1 SINE in clones, and clone-specific effects, as reflected by increased variability in the methylation of the PRE-1 SINE.
Severely affected clones are very likely to die in utero, whereas those that survive to birth, such as the cloned pigs used in this study, show a greater degree of variation in susceptible traits than expected for genetically identical animals. Overall, our results indicate that although cloning creates animals within the normal phenotypic range, it does affect some traits by increasing variability associated with that phenotype. This clonal variability may be useful for mapping epigenetically modified traits, either imprinted modifiers or genes susceptible to methylation effects. More importantly, the epigenetic disregulation introduced by the cloning process must be taken into account when considering the utilization of this technology for the generation of human stem cells, for the generation of companion animal clones, and for the generation of clones for experimental studies with the expectation of reduced genotypic and phenotypic variability among experimental animals.