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.
Project description:The mixing of mitochondrial DNA (mtDNA) from the donor cell and the recipient oocyte in embryos and offspring derived from somatic cell nuclear transfer (SCNT) compromises genetic integrity and affects embryo development. We set out to generate SCNT embryos that inherited their mtDNA from the recipient oocyte only, as is the case following natural conception. While SCNT blastocysts produced from Holstein (Bos taurus) fibroblasts depleted of their mtDNA, and oocytes derived from Angus (Bos taurus) cattle possessed oocyte mtDNA only, the coexistence of donor cell and oocyte mtDNA resulted in blastocysts derived from nondepleted cells. Moreover, the use of the reprogramming agent, Trichostatin A (TSA), further improved the development of embryos derived from depleted cells. RNA-seq analysis highlighted 35 differentially expressed genes from the comparison between blastocysts generated from nondepleted cells and blastocysts from depleted cells, both in the presence of TSA. The only differences between these two sets of embryos were the presence of donor cell mtDNA, and a significantly higher mtDNA copy number for embryos derived from nondepleted cells. Furthermore, the use of TSA on embryos derived from depleted cells positively modulated the expression of CLDN8, TMEM38A, and FREM1, which affect embryonic development. In conclusion, SCNT embryos produced by mtDNA depleted donor cells have the same potential to develop to the blastocyst stage without the presumed damaging effect resulting from the mixture of donor and recipient mtDNA.
Project description:Mitochondrial DNA (mtDNA) segregation associated with donor-recipient mtDNA mismatch in mitochondria replacement therapy leads to unknown risks. Here, to explore whether matching mtDNA haplotypes contributes to ameliorating segregation, we reproduced various degrees of heteroplasmic mice with three single nucleotide polymorphisms to monitor segregation severity. "Segregation" presented in tissues of heteroplasmic mice containing low-level donor mtDNA heteroplasmy, and disappeared as donor mtDNA heteroplasmy levels ascended. Meanwhile, we found that distribution of donor mtDNA among the blastomeres of preimplantation embryos from the heteroplasmic mice shared the same tendency as that in adult tissues. Statistical analysis showed that no selective replication of donor mtDNA occurred during lifespan. Tracking donor mtDNA distribution showed that uneven distribution of donor mtDNA among embryonic blastomeres gradually became even as donor mtDNA heteroplasmy increased, indicating that the "segregation" in tissues was inherited from the uneven distribution. Our finding suggested that donor-recipient mtDNA matching could circumvent segregation in mitochondria replacement therapy.
Project description:Somatic cell nuclear transfer (SCNT) is a key technology with broad applications that range from production of cloned farm animals to derivation of patient-matched stem cells or production of humanized animal organs for xenotransplantation. However, effects of aberrant epigenetic reprogramming on gene expression compromise cell and organ phenotype, resulting in low success rate of SCNT. Standard SCNT procedures include enucleation of recipient oocytes before the nuclear donor cell is introduced. Enucleation removes not only the spindle apparatus and chromosomes of the oocyte but also the perinuclear, mitochondria rich, ooplasm. Here, we use a Bos taurus SCNT model with in vitro fertilized (IVF) and in vivo conceived controls to demonstrate a ∼50% reduction in mitochondrial DNA (mtDNA) in the liver and skeletal muscle, but not the brain, of SCNT fetuses at day 80 of gestation. In the muscle, we also observed significantly reduced transcript abundances of mtDNA-encoded subunits of the respiratory chain. Importantly, mtDNA content and mtDNA transcript abundances correlate with hepatomegaly and muscle hypertrophy of SCNT fetuses. Expression of selected nuclear-encoded genes pivotal for mtDNA replication was similar to controls, arguing against an indirect epigenetic nuclear reprogramming effect on mtDNA amount. We conclude that mtDNA depletion is a major signature of perturbations after SCNT. We further propose that mitochondrial perturbation in interaction with incomplete nuclear reprogramming drives abnormal epigenetic features and correlated phenotypes, a concept supported by previously reported effects of mtDNA depletion on the epigenome and the pleiotropic phenotypic effects of mtDNA depletion in humans. This provides a novel perspective on the reprogramming process and opens new avenues to improve SCNT protocols for healthy embryo and tissue development.
Project description:We investigated the mitochondrial DNA (mtDNA) composition in one of the largest adult somatic mammalian clones (n = 20) reported so far. The healthy cloned cattle were derived from nuclear transfer of an identical nuclear genetic background (mural granulosa donor cells including surrounding cytoplasm) into enucleated oocytes with either Bos indicus or B. taurus mtDNA. Here we report the first cases of coexisting mtDNAs of two closely related subspecies following nuclear transfer. Heteroplasmy (0.6-2.8%) was found in 4 out of 11 cross-subspecies cloned cattle. Quantitation was performed using "amplification refractory mutation system (ARMS) allele-specific real-time PCR." We determined that the ratio of donor cell to recipient cytoplast mtDNA copy number was 0.9% before nuclear transfer. Therefore, we concluded that the percentage of donor cell mtDNA in the heteroplasmic intersubspecific cloned animals is in accordance with neutral transmission of donor mtDNA. We determined an amino acid sequence divergence of up to 1.3% for the two subspecies-specific mtDNA haplotypes. In addition, intrasubspecific B. indicus heteroplasmy of approximately 1% (but up to 7.3 and 12.7% in muscle and follicular cells of one animal) was detected in 7 out of the 9 B. indicus intrasubspecific cloned cattle.
Project description:Transvaginal ultrasound-guided follicle aspiration is one method of obtaining recipient oocytes for equine somatic cell nuclear transfer (SCNT). This study was conducted: (1) to evaluate the possibility of oocyte aspiration from pre-ovulatory follicles using a short disposable needle system (14-G) by comparing the oocyte recovery rate with that of a long double lumen needle (12-G); (2) to investigate the developmental competence of recovered oocytes after SCNT and embryo transfer. The recovery rates with the short disposable needle vs. the long needle were not significantly different (47.5% and 35.0%, respectively). Twenty-six SCNT embryos were transferred to 13 mares, and one mare delivered a live offspring at Day 342. There was a perfect identity match between the cloned foal and the cell donor after analysis of microsatellite DNA, and the mitochondrial DNA of the cloned foal was identical with that of the oocyte donor. These results demonstrated that the short disposable needle system can be used to recover oocytes to use as cytoplasts for SCNT, in the production of cloned foals and for other applications in equine embryology.
Project description:We generated cattle embryos using mitochondrial supplementation and somatic cell nuclear transfer (SCNT), named miNT, to determine how additional mitochondrial DNA (mtDNA) modulates the nuclear genome. To eliminate any confounding effects from somatic cell mtDNA in intraspecies SCNT, donor cell mtDNA was depleted prior to embryo production. Additional oocyte mtDNA did not affect embryo development rates but increased mtDNA copy number in blastocyst stage embryos. Moreover, miNT-derived blastocysts had different gene expression profiles when compared with SCNT-derived blastocysts. Additional mtDNA increased expression levels of genes involved in oxidative phosphorylation, cell cycle and DNA repair. Supplementing the embryo culture media with a histone deacetylase inhibitor, Trichostatin A (TSA), had no beneficial effects on the development of miNT-derived embryos, unlike SCNT-derived embryos. When compared with SCNT-derived blastocysts cultured in the presence of TSA, additional mtDNA alone had beneficial effects as the activity of glycolysis may increase and embryonic cell death may decrease. However, these beneficial effects were not found with additional mtDNA and TSA together, suggesting that additional mtDNA alone enhances reprogramming. In conclusion, additional mtDNA increased mtDNA copy number and expression levels of genes involved in energy production and embryo development in blastocyst stage embryos emphasising the importance of nuclear-mitochondrial interactions.
Project description:Somatic cell nuclear transfer (SCNT) offers great potential for developing better animal models of human disease. The domestic ferret (Mustela putorius furo) is an ideal animal model for influenza infections and potentially other human respiratory diseases such as cystic fibrosis, where mouse models have failed to reproduce the human disease phenotype. Here, we report the successful production of live cloned, reproductively competent, ferrets using species-specific SCNT methodologies. Critical to developing a successful SCNT protocol for the ferret was the finding that hormonal treatment, normally used for superovulation, adversely affected the developmental potential of recipient oocytes. The onset of Oct4 expression was delayed and incomplete in parthenogenetically activated oocytes collected from hormone-treated females relative to oocytes collected from females naturally mated with vasectomized males. Stimulation induced by mating and in vitro oocyte maturation produced the optimal oocyte recipient for SCNT. Although nuclear injection and cell fusion produced mid-term fetuses at equivalent rates (approximately 3-4%), only cell fusion gave rise to healthy surviving clones. Single cell fusion rates and the efficiency of SCNT were also enhanced by placing two somatic cells into the perivitelline space. These species-specific modifications facilitated the birth of live, healthy, and fertile cloned ferrets. The development of microsatellite genotyping for domestic ferrets confirmed that ferret clones were genetically derived from their respective somatic cells and unrelated to their surrogate mother. With this technology, it is now feasible to begin generating genetically defined ferrets for studying transmissible and inherited human lung diseases. Cloning of the domestic ferret may also aid in recovery and conservation of the endangered black-footed ferret and European mink.
Project description:Mitochondrial diseases display pathological phenotypes according to the mixture of mutant versus wild-type mitochondrial DNA (mtDNA), known as heteroplasmy. We herein examined the impact of nuclear reprogramming and clonal isolation of induced pluripotent stem cells (iPSC) on mitochondrial heteroplasmy. Patient-derived dermal fibroblasts with a prototypical mitochondrial deficiency diagnosed as mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) demonstrated mitochondrial dysfunction with reduced oxidative reserve due to heteroplasmy at position G13513A in the ND5 subunit of complex I. Bioengineered iPSC clones acquired pluripotency with multilineage differentiation capacity and demonstrated reduction in mitochondrial density and oxygen consumption distinguishing them from the somatic source. Consistent with the cellular mosaicism of the original patient-derived fibroblasts, the MELAS-iPSC clones contained a similar range of mtDNA heteroplasmy of the disease-causing mutation with identical profiles in the remaining mtDNA. High-heteroplasmy iPSC clones were used to demonstrate that extended stem cell passaging was sufficient to purge mutant mtDNA, resulting in isogenic iPSC subclones with various degrees of disease-causing genotypes. On comparative differentiation of iPSC clones, improved cardiogenic yield was associated with iPSC clones containing lower heteroplasmy compared with isogenic clones with high heteroplasmy. Thus, mtDNA heteroplasmic segregation within patient-derived stem cell lines enables direct comparison of genotype/phenotype relationships in progenitor cells and lineage-restricted progeny, and indicates that cell fate decisions are regulated as a function of mtDNA mutation load. The novel nuclear reprogramming-based model system introduces a disease-in-a-dish tool to examine the impact of mutant genotypes for MELAS patients in bioengineered tissues and a cellular probe for molecular features of individual mitochondrial diseases.
Project description:Determination mitochondrial DNA (mtDNA) sequences from extremely small amounts of DNA extracted from tissue of limited amounts and/or degraded samples is frequently employed in medical, forensic, and anthropologic studies. Polymerase chain reaction (PCR) amplification followed by DNA cloning is a routine method, especially to examine heteroplasmy of mtDNA mutations. In this review, we compare the mtDNA mutation patterns detected by three different sequencing strategies. Cloning and sequencing methods that are based on PCR amplification of DNA extracted from either single cells or pooled cells yield a high frequency of mutations, partly due to the artifacts introduced by PCR and/or the DNA cloning process. Direct sequencing of PCR product which has been amplified from DNA in individual cells is able to detect the low levels of mtDNA mutations present within a cell. We further summarize the findings in our recent studies that utilized this single cell method to assay mtDNA mutation patterns in different human blood cells. Our data show that many somatic mutations observed in the end-stage differentiated cells are found in hematopoietic stem cells (HSCs) and progenitors within the CD34(+) cell compartment. Accumulation of mtDNA variations in the individual CD34+ cells is affected by both aging and family genetic background. Granulocytes harbor higher numbers of mutations compared with the other cells, such as CD34(+) cells and lymphocytes. Serial assessment of mtDNA mutations in a population of single CD34(+) cells obtained from the same donor over time suggests stability of some somatic mutations. CD34(+) cell clones from a donor marked by specific mtDNA somatic mutations can be found in the recipient after transplantation. The significance of these findings is discussed in terms of the lineage tracing of HSCs, aging effect on accumulation of mtDNA mutations and the usage of mtDNA sequence in forensic identification.
Project description:Somatic cell nuclear transfer (SCNT) is a well-known laboratory technique. The principle of the SCNT involves the reprogramming a somatic nucleus by injecting a somatic cell into a recipient oocyte whose nucleus has been removed. Therefore, the nucleus donor cells are considered as a crucial factor in SCNT. Cell cycle synchronization of nucleus donor cells at G0/G1 stage can be induced by contact inhibition or serum starvation. In this study, acteoside, a phenylpropanoid glycoside compound, was investigated to determine whether it is applicable for inducing cell cycle synchronization, cytoprotection, and improving SCNT efficiency in canine fetal fibroblasts. Primary canine fetal fibroblasts were treated with acteoside (10, 30, 50 ?M) for various time periods (24, 48 and 72 hours). Cell cycle synchronization at G0/G1 stage did not differ significantly with the method of induction: acteoside treatment, contact inhibition or serum starvation. However, of these three treatments, serum starvation resulted in significantly increased level of reactive oxygen species (ROS) (99.5 ± 0.3%) and apoptosis. The results also revealed that acteoside reduced ROS and apoptosis processes including necrosis in canine fetal fibroblasts, and improved the cell survival. Canine fetal fibroblasts treated with acteoside were successfully arrested at the G0/G1 stage. Moreover, the reconstructed embryos using nucleus donor cells treated with acteoside produced a healthy cloned dog, but not the embryos produced using nucleus donor cells subjected to contact inhibition. In conclusion, acteoside induced cell cycle synchronization of nucleus donor cells would be an alternative method to improve the efficiency of canine SCNT because of its cytoprotective effects.