Mitochondrial DNA heteroplasmy in ovine fetuses and sheep cloned by somatic cell nuclear transfer

Abstract

Background: The mitochondrial DNA (mtDNA) of the cloned sheep "Dolly" and nine other ovine clones produced by somatic cell nuclear transfer (SCNT) was reported to consist only of recipient oocyte mtDNA without any detectable mtDNA contribution from the nucleus donor cell. In cattle, mouse and pig several or most of the clones showed transmission of nuclear donor mtDNA resulting in mitochondrial heteroplasmy. To clarify the discrepant transmission pattern of donor mtDNA in sheep clones we analysed the mtDNA composition of seven fetuses and five lambs cloned from fetal fibroblasts.

Results: The three fetal fibroblast donor cells used for SCNT harboured low mtDNA copy numbers per cell (A: 753 +/- 54, B: 292 +/- 33 and C: 561 +/- 88). The ratio of donor to recipient oocyte mtDNAs was determined using a quantitative amplification refractory mutation system (ARMS) PCR (i.e. ARMS-qPCR). For quantification of SNP variants with frequencies below 0.1% we developed a restriction endonuclease-mediated selective quantitative PCR (REMS-qPCR). We report the first cases (n = 4 fetuses, n = 3 lambs) of recipient oocyte/nuclear donor mtDNA heteroplasmy in SCNT-derived ovine clones demonstrating that there is no species-effect hindering ovine nucleus-donor mtDNA from being transmitted to the somatic clonal offspring. Most of the heteroplasmic clones exhibited low-level heteroplasmy (0.1% to 0.9%, n = 6) indicating neutral transmission of parental mtDNAs. High-level heteroplasmy (6.8% to 46.5%) was observed in one case. This clone possessed a divergent recipient oocyte-derived mtDNA genotype with three rare amino acid changes compared to the donor including one substitution at an evolutionary conserved site.

Conclusion: Our study using state-of-the-art techniques for mtDNA quantification, like ARMS-qPCR and the novel REMS-qPCR, documents for the first time the transmission of donor mtDNA into somatic sheep clones. MtDNA heteroplasmy was detected in seven of 12 clones tested, whereby all but one case revealed less than 1% mtDNA contribution from the nuclear donor cell suggesting neutral segregation.

Figure 1

The majority of the clonal offspring harbours different recipient oocyte-derived mitochondrial genotypes. An unrooted phylogenetic tree derived by maximum likelihood using TREE-PUZZLE 5.2 with default settings from an alignment of concatenated mtDNA sequences (3806 nucleotides: control region, MT-CO1, MT-CO3, and MT-ND4L) of the three donor cell lines, their SCNT clones (CA1 to CA7, CB1 to CB3, and CC1 and CC2) and of an European mitochondrial reference genome (GenBank:AF010406). The scale bar represents 0.001 substitutions per site, and quartet puzzling values are shown (all are >50). The numbers at the nodes (quartet puzzling values) indicate the frequencies of occurrence for 1,000 replicate trees. Quartet puzzling support values provide an estimate of support of a given branch and can be interpreted in much the same way as bootstrap values. CA5 is the most divergent donor A-derived clone which is highlighted by the ellipse (see below for amino acid changes).

Figure 2

The novel REMS-qPCR improves the quantitative detection limit for low-abundant point mutations. This method requires pre-PCR cleavage of the high-abundant variant (non-target DNA here: recipient-oocyte mtDNA) and ARMS-qPCR (non-discriminative (ND) and discriminative (D) assays) for the low-abundant SNP variant (target here: donor mtDNA) at the mutated site. The amplification efficiency of the D assay was 91%. Without further optimization concerning conditions for enzymatic digestion, quantification by REMS-qPCR targeting a single donor-B-specific SNP reached a detection limit of 0.02%, i.e. a point mutation discrimination selectivity factor of 5 × 10³. It allowed detection of heteroplasmy (0.1%) in the donor B-derived clone CB2. This could not be detected by conventional ARMS-qPCR discriminating point mutations only down to 0.1% (see: illustrated improvement of discrimination). For clarity each plot is presented as the mean calculated from duplicate amplification reactions. Individual Ct values, i.e. PCR cycle numbers at which plots crossed an arbitrarily placed signal threshold, are given in the figure key. An independent technical replicate of this REMS-qPCR experiment demonstrated reproducibility of the method (data not shown).