Mitochondrial DNA heteroplasmy in cloned cattle produced by fetal and adult cell cloning

Abstract

Mammals have been cloned from adult donor cells. Here we report the first cases of mitochondrial DNA (mtDNA) heteroplasmy in adult mammalian clones generated from fetal and adult donor cells. The heteroplasmic clones included a healthy cattle equivalent of the sheep Dolly, for which a lack of heteroplasmy was reported.

The effect of mitochondrial genome on architectural remodeling and epigenetic reprogramming of donor cell nuclei in mammalian nuclear transfer-derived embryos

“…That is why calves derived from NT blastocysts transplanted into surrogate mothers, exhibited mtDNA heteroplasmy, induced by both cellular mosaicism of mitochondrial genome sequences and neutral (random) segregation of parental mtDNA haplotypes (the so-called mitotypes) during early embryogenesis (Steinborn et al, 1998b;Hiendleder et al, 1999;Takeda et al, 2003;Figure 3). Recently, Steinborn et al (2000) reported mitochondrial heteroplasmy in cloned cattle generated from Figure 3. Schematic diagram of intraspecies somatic cloning (nuclear transfer of allogeneic somatic cells).…”

“…Thus far, the fate of foreign mtDNAs is still controversial in intraspecies cloned embryos. In the bovine nuclear transferred embryos, mitochondrial genome primarily arises from the recipient oocytes (Do et al, 2001;Meirelles et al, 2001), whereas in the others mtDNA copies appear to be heteroplasmic (Steinborn et al, 2000(Steinborn et al, , 2002Do et al, 2002;Hiendleder et al, 2003;Takeda et al, 2003) SAMIEC M.…”

“…PCR-mediated restriction fragment length polymorphism (PCR-RFLP) in cloned sheep (Evans et al, 1999) and cloned bovine foetuses (Hiendleder et al, 2004a,b); 3. allele-specific PCR (AS-PCR) in cloned bovine embryos and calves (Steinborn et al, 1998b;Do et al, 2002); 4. direct PCR product sequencing in interspecies cloned panda-rabbit embryos (Chen et al, 2002) and macaquerabbit embryos , cloned bovine embryos (Do et al, 2002) and ooplasm-transferred humans (Brenner et al, 2000;Barritt et al, 2001); 5. DNA chromatography in cloned bovine embryos (Do et al, 2002) and ooplasmtransferred humans (Brenner et al, 2000;Barritt et al, 2001), and 6. allele-specific real-time PCR in cloned cattle (Steinborn et al, 2000) or real-time (rapid cycle fluorescence monitored) PCR in interspecies cloned macaque-rabbit embryos (Yang et al, 2004). Of these methods, allele-specific PCR is considered to be the most sensitive and suitable technique for the analysis of the mtDNA distribution patterns in the pre-and postimplanted NT-reconstituted embryos.…”

“…Thus, the real-time PCR method with sensitivity to detect extremely low levels of mitochondrial genome heteroplasmy (less than ten copies of mtDNA) can also be extended to identification of the nuclear genes. Steinborn et al (2000) quantified the percentage of somatic cell-transmitted mtDNA fractions in cloned bovine embryos and born calves by allele-specific real-time PCR. It was revealed that the ratio of nuclear donor cell-descended mtDNAs to recipient ooplast-inherited mitotypes was approximately 2-5% (Steinborn et al, 2000) and less than 3-4% (Takeda et al, 2003) in the reconstructed bovine 1-cell embryos immediately after electrofusion of somatic cell-cytoplast complexes.…”

“…Steinborn et al (2000) quantified the percentage of somatic cell-transmitted mtDNA fractions in cloned bovine embryos and born calves by allele-specific real-time PCR. It was revealed that the ratio of nuclear donor cell-descended mtDNAs to recipient ooplast-inherited mitotypes was approximately 2-5% (Steinborn et al, 2000) and less than 3-4% (Takeda et al, 2003) in the reconstructed bovine 1-cell embryos immediately after electrofusion of somatic cell-cytoplast complexes. In the experiments by Steinborn et al (2000) it was shown that the level of somatogenic mtDNA copies ranged from 0.4 to 4% and remained the same throughout all the pre-and postimplantation development of bovine nuclear transfer embryos.…”