Aberrant heteroplasmic transmission of mtDNA in cloned pigs arising from double nuclear transfer

Abstract

Double nuclear transfer begins with the transfer of nuclear DNA from a donor cell into an enucleated recipient oocyte. This reconstructed oocyte is allowed to develop to the pronuclear stage, where the pronuclei are transferred into an enucleated zygote. This reconstructed zygote is then transferred to a surrogate sow. The genetic integrity of cloned offspring can be compromised by the transmission of mitochondrial DNA from the donor cell, the recipient oocyte and the recipient zygote. We have verified through the use of sequence analysis, restriction fragment length polymorphism analysis, allele specific PCR and primer extension polymorphism analysis that following double nuclear transfer the donor cell mtDNA is eliminated. However, it is likely that the recipient oocyte and zygote mitochondrial DNA are transmitted to the offspring, indicating bimaternal mitochondrial DNA transmission. This pattern of mtDNA inheritance is similar to that observed following cytoplasmic transfer and violates the strict unimaternal inheritance of mitochondrial DNA to offspring. This form of transmission raises concerns regarding the genetic integrity of cloned offspring and their uses in studies that require metabolic analysis or a stable genetic environment where only one variable is under analysis, such as in knockout technology.

Interspecies Somatic Cell Nuclear Transfer Is Dependent on Compatible Mitochondrial DNA and Reprogramming Factors

“…To this extent, the mixing of mtDNA genomes from different breeds within a species induces mild transitions in sequence variation between the coding genes. This is reflected in interspecific bovine [43] and porcine [24] SCNT offspring where the sequence variants between the donor cell and recipient oocyte mtDNA populations result in different amino acids being generated within one offspring. These offspring seem to survive parturition though in many cases such combinations fail to survive the first six months post-natally [44].…”

“…For example, donor cell mtDNA has been detected in bovine embryos derived by both intra- [21] and inter-specific NT [18], though not in all cases [18], [19], and in caprine embryos [22] and porcine offspring [23] derived by interspecific SCNT. However, as there are sequence variations in the mtDNA coding genes for breeds within the same species, this can result in different combinations of amino acid synthesis and the degree of heteroplasmy could considerably reduce the ability of any resultant stem cells to generate sufficient ATP through OXPHOS [24].…”

The Relationship Between Pluripotency and Mitochondrial DNA Proliferation During Early Embryo Development and Embryonic Stem Cell Differentiation

“…Differences in mtDNA sequence between donor cell and recipient oocyte are likely to give rise to proteins with slight differences in their respective amino acid sequences [185]. This is likely to result in inadequate interaction between the individual ETC subunits which might lead to reduced oxidative capacity and consequently embryonic or fetal death [187,188]. In addition, failure or defective interaction of the donor nDNA-encoded mtDNA transcription and replication factors with the recipient oocyte's mtDNA would result in compromised mtDNA transcription and replication and most likely lead to embryonic arrest or to the appearance of mtDNAdepletion syndromes after birth.…”

Metabolomic phenotyping of a cloned pig model

“…Cloned pigs will have identical DNA sequences and in principle identical phenotypes. However, in the cloning process, single somatic cell nuclei are introduced into each their enucleated oocyte containing mitochondrial DNA, so a small subset of mitochondrial proteins will be of maternal origin [8]. Additionally, the somatic cell nuclei may have different epigenetic constitution, i.e.…”