Characterization of a donor mitochondrial DNA transmission bottleneck in nuclear transfer derived cow lineages

Abstract

In embryos derived by nuclear-transfer (NT), fusion of donor cells with recipient oocytes resulted in varying patterns of mitochondrial DNA (mtDNA) transmission in NT animals. Distribution of donor cell mtDNA (D-mtDNA) found in offspring of NT-derived founders may also vary from donor cell and host embryo heteroplasmy to host embryo homoplasmy. Here we examined the transmission of mtDNA from NT cows to G(1) offspring. Eleven NT founder cows were produced by fusion of enucleated oocytes (Holstein/Japanese Black) with Jersey/ Holstein oviduct epithelial cells, or Holstein/Japanese Black cumulus cells. Transmission of mtDNA was analyzed by PCR mediated single-strand conformation polymorphism of the D-loop region. In six of seven animals sampled postmortem, heteroplasmy were detected in various tissues, while D-mtDNA could not be detected in blood or hair samples from four live animals. The average proportion of D-mtDNA detected in one NT cow was 7.6%, and those in other cows were <5%. Heteroplasmic NT cows (n = 6) generated a total 12 G(1) offspring. Four of 12 G(1) offspring exhibited high percentages of D-mtDNA populations (range 17-51%). The remaining eight G(1) offspring had slightly or undetectable D-mtDNA (<5%). Generally, a genetic bottleneck in the female germ-line should favor a homoplasmic state. However, proportions of some G(1) offspring maintained heteroplasmy with a much higher percentage of D-mtDNA than their NT dams, which may also reflect a segregation distortion caused by the proposed mitochondrial bottleneck. These results demonstrate that D-mtDNA in NT cows is transmitted to G(1) offspring with varying efficiencies.

Pronounced Segregation of Donor Mitochondria Introduced by Bovine Ooplasmic Transfer to the Female Germ-Line1

“…In higher mammals, the segregation of donor mtDNA through the germ-line bottleneck was assessed previously only indirectly, using tissues of a few SCNT offspring [47].…”

Mitochondrial DNA transmission and confounding mitochondrial influences in cloned cattle and pigs

“…Although most bovine SCNT offspring show mtDNA genotypes that differ from those of the targeted (founder) animals ( Fig. 1) [9,10], nuclear transfer is regarded as the most effective technique to exchange mtDNA in a single generation [4,11]. The main reason for mitochondrial respiration defects in mitochondrial diseases is accumulation of pathogenic mutant mtDNAs; therefore, nuclear transfer has been considered the most effective treatment strategy to preferentially eliminate mutant mtDNA molecules or to dilute the molecules to below a threshold proportion for the expression of disease phenotypes.…”

“…CA3 and CA4 exhibited a mixture of genotypes with D-mtDNA [79] more sequence variants in a cell, a genetic bottleneck in the female germ line will generally favor the transmission of a single mitochondrial population (homoplasmy) [13,27,28]. However, in SCNT animals both stable and unstable transmission of D-mtDNA was found in porcine and bovine G n s [10,17]. In our study, D-mtDNA was present at ranges of 0.1-1.0 % of total mtDNA in SCNT pigs derived from Chinese pig (Meishan breed) fibroblasts microinjected into European pig oocytes (maternal Landrace breed) [29].…”

Further Advice on the Implications of Animal Cloning (SCNT)

“…It has been demonstrated that donor mitochondrial DNA (mtDNA) is transmitted to clone offspring with varying efficiencies (Takeda et al, 2008). Four cows (F1) with mtDNA heteroplasmy showed normal growth, productivity and lactation characteristics and productivity.…”