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1.
Biol Reprod ; 102(3): 607-619, 2020 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-31621839

RESUMO

Heritable mitochondrial DNA (mtDNA) mutations are common, yet only a few recurring pathogenic mtDNA variants account for the majority of known familial cases in humans. Purifying selection in the female germline is thought to be responsible for the elimination of most harmful mtDNA mutations during oogenesis. Here we show that deleterious mtDNA mutations are abundant in ovulated mature mouse oocytes and preimplantation embryos recovered from PolG mutator females but not in their live offspring. This implies that purifying selection acts not in the maternal germline per se, but during post-implantation development. We further show that oocyte mtDNA mutations can be captured and stably maintained in embryonic stem cells and then reintroduced into chimeras, thereby allowing examination of the effects of specific mutations on fetal and postnatal development.

4.
PLoS One ; 13(7): e0201304, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30040856

RESUMO

The accumulation of acquired mitochondrial genome (mtDNA) mutations with aging in somatic cells has been implicated in mitochondrial dysfunction and linked to age-onset diseases in humans. Here, we asked if somatic mtDNA mutations are also associated with aging in the mouse. MtDNA integrity in multiple organs and tissues in young and old (2-34 months) wild type (wt) mice was investigated by whole genome sequencing. Remarkably, no acquired somatic mutations were detected in tested tissues. However, we identified several non-synonymous germline mtDNA variants whose heteroplasmy levels (ratio of normal to mutant mtDNA) increased significantly with aging suggesting clonal expansion of inherited mtDNA mutations. Polg mutator mice, a model for premature aging, exhibited both germline and somatic mtDNA mutations whose numbers and heteroplasmy levels increased significantly with age implicating involvement in premature aging. Our results suggest that, in contrast to humans, acquired somatic mtDNA mutations do not accompany the aging process in wt mice.


Assuntos
Envelhecimento , DNA Mitocondrial/genética , Camundongos/genética , Mutação , Senilidade Prematura/genética , Senilidade Prematura/veterinária , Animais , Polimerase do DNA Mitocondrial/genética , Feminino , Células Germinativas/metabolismo , Masculino , Camundongos/embriologia , Camundongos/fisiologia , Camundongos Endogâmicos C57BL , Mitocôndrias/genética
5.
Nature ; 548(7668): 413-419, 2017 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-28783728

RESUMO

Genome editing has potential for the targeted correction of germline mutations. Here we describe the correction of the heterozygous MYBPC3 mutation in human preimplantation embryos with precise CRISPR-Cas9-based targeting accuracy and high homology-directed repair efficiency by activating an endogenous, germline-specific DNA repair response. Induced double-strand breaks (DSBs) at the mutant paternal allele were predominantly repaired using the homologous wild-type maternal gene instead of a synthetic DNA template. By modulating the cell cycle stage at which the DSB was induced, we were able to avoid mosaicism in cleaving embryos and achieve a high yield of homozygous embryos carrying the wild-type MYBPC3 gene without evidence of off-target mutations. The efficiency, accuracy and safety of the approach presented suggest that it has potential to be used for the correction of heritable mutations in human embryos by complementing preimplantation genetic diagnosis. However, much remains to be considered before clinical applications, including the reproducibility of the technique with other heterozygous mutations.


Assuntos
Proteínas de Transporte/genética , Embrião de Mamíferos/metabolismo , Edição de Genes/métodos , Mutação/genética , Adulto , Alelos , Blastocisto/metabolismo , Blastocisto/patologia , Divisão Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Quebras de DNA de Cadeia Dupla , Embrião de Mamíferos/patologia , Marcação de Genes , Teste de Complementação Genética , Heterozigoto , Homozigoto , Humanos , Masculino , Mosaicismo , Reparo de DNA por Recombinação/genética , Fase S , Moldes Genéticos , Zigoto/metabolismo , Zigoto/patologia
6.
Cell Stem Cell ; 20(1): 112-119, 2017 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-27840020

RESUMO

Oocyte defects lie at the heart of some forms of infertility and could potentially be addressed therapeutically by alternative routes for oocyte formation. Here, we describe the generation of functional human oocytes following nuclear transfer of first polar body (PB1) genomes from metaphase II (MII) oocytes into enucleated donor MII cytoplasm (PBNT). The reconstructed oocytes supported the formation of de novo meiotic spindles and, after fertilization with sperm, meiosis completion and formation of normal diploid zygotes. While PBNT zygotes developed to blastocysts less frequently (42%) than controls (75%), genome-wide genetic, epigenetic, and transcriptional analyses of PBNT and control ESCs indicated comparable numbers of structural variations and markedly similar DNA methylation and transcriptome profiles. We conclude that rescue of PB1 genetic material via introduction into donor cytoplasm may offer a source of oocytes for infertility treatment or mitochondrial replacement therapy for mtDNA disease.


Assuntos
Genoma Humano , Técnicas de Transferência Nuclear , Oócitos/metabolismo , Corpos Polares/metabolismo , Adulto , Blastocisto/metabolismo , Metilação de DNA/genética , Desenvolvimento Embrionário/genética , Epigênese Genética , Feminino , Fertilização In Vitro , Perfilação da Expressão Gênica , Instabilidade Genômica , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Masculino , Metáfase , Ploidias , Análise de Sequência de RNA , Espermatozoides/metabolismo , Fuso Acromático/metabolismo , Transcrição Genética
7.
Nature ; 540(7632): 270-275, 2016 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-27919073

RESUMO

Maternally inherited mitochondrial (mt)DNA mutations can cause fatal or severely debilitating syndromes in children, with disease severity dependent on the specific gene mutation and the ratio of mutant to wild-type mtDNA (heteroplasmy) in each cell and tissue. Pathogenic mtDNA mutations are relatively common, with an estimated 778 affected children born each year in the United States. Mitochondrial replacement therapies or techniques (MRT) circumventing mother-to-child mtDNA disease transmission involve replacement of oocyte maternal mtDNA. Here we report MRT outcomes in several families with common mtDNA syndromes. The mother's oocytes were of normal quality and mutation levels correlated with those in existing children. Efficient replacement of oocyte mutant mtDNA was performed by spindle transfer, resulting in embryos containing >99% donor mtDNA. Donor mtDNA was stably maintained in embryonic stem cells (ES cells) derived from most embryos. However, some ES cell lines demonstrated gradual loss of donor mtDNA and reversal to the maternal haplotype. In evaluating donor-to-maternal mtDNA interactions, it seems that compatibility relates to mtDNA replication efficiency rather than to mismatch or oxidative phosphorylation dysfunction. We identify a polymorphism within the conserved sequence box II region of the D-loop as a plausible cause of preferential replication of specific mtDNA haplotypes. In addition, some haplotypes confer proliferative and growth advantages to cells. Hence, we propose a matching paradigm for selecting compatible donor mtDNA for MRT.


Assuntos
DNA Mitocondrial/genética , DNA Mitocondrial/uso terapêutico , Herança Materna/genética , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Terapia de Substituição Mitocondrial/métodos , Mutação , Oócitos/metabolismo , Blastocisto/citologia , Blastocisto/metabolismo , Linhagem Celular , Sequência Conservada/genética , DNA Mitocondrial/biossíntese , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Haplótipos/genética , Humanos , Masculino , Meiose , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/prevenção & controle , Doação de Oócitos , Oócitos/citologia , Oócitos/patologia , Fosforilação Oxidativa , Linhagem , Polimorfismo Genético
8.
Cell Metab ; 24(2): 283-94, 2016 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-27425585

RESUMO

Vertebrate cells carry two different genomes, nuclear (nDNA) and mitochondrial (mtDNA), both encoding proteins involved in oxidative phosphorylation. Because of the extensive interactions, adaptive coevolution of the two genomes must occur to ensure normal mitochondrial function. To investigate whether incompatibilities between these two genomes could contribute to interspecies reproductive barriers, we performed reciprocal mtDNA replacement (MR) in zygotes between widely divergent Mus m. domesticus (B6) and conplastic Mus m. musculus (PWD) mice. Transfer of MR1 cybrid embryos (B6nDNA-PWDmtDNA) supported normal development of F1 offspring with reduced male fertility but unaffected reproductive fitness in females. Furthermore, donor PWD mtDNA was faithfully transmitted through the germline into F2 and F3 generations. In contrast, reciprocal MR2 (PWDnDNA-B6mtDNA) produced high embryonic loss and stillborn rates, suggesting an association between mitochondrial function and infertility. These results strongly suggest that functional incompatibility between nuclear and mitochondrial genomes contributes to interspecies reproductive isolation in mammals.


Assuntos
Núcleo Celular/genética , Genoma Mitocondrial , Reprodução/genética , Animais , Blastocisto/metabolismo , Cruzamentos Genéticos , DNA Mitocondrial/genética , Perda do Embrião/genética , Desenvolvimento Embrionário/genética , Feminino , Células Germinativas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Especificidade da Espécie
9.
Cell Stem Cell ; 18(5): 625-36, 2016 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-27151456

RESUMO

The genetic integrity of iPSCs is an important consideration for therapeutic application. In this study, we examine the accumulation of somatic mitochondrial genome (mtDNA) mutations in skin fibroblasts, blood, and iPSCs derived from young and elderly subjects (24-72 years). We found that pooled skin and blood mtDNA contained low heteroplasmic point mutations, but a panel of ten individual iPSC lines from each tissue or clonally expanded fibroblasts carried an elevated load of heteroplasmic or homoplasmic mutations, suggesting that somatic mutations randomly arise within individual cells but are not detectable in whole tissues. The frequency of mtDNA defects in iPSCs increased with age, and many mutations were non-synonymous or resided in RNA coding genes and thus can lead to respiratory defects. Our results highlight a need to monitor mtDNA mutations in iPSCs, especially those generated from older patients, and to examine the metabolic status of iPSCs destined for clinical applications.


Assuntos
Envelhecimento/genética , DNA Mitocondrial/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação/genética , Adulto , Idoso , Células Sanguíneas/metabolismo , Fibroblastos/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Pele/citologia
10.
Nature ; 524(7564): 234-8, 2015 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-26176921

RESUMO

Mitochondria have a major role in energy production via oxidative phosphorylation, which is dependent on the expression of critical genes encoded by mitochondrial (mt)DNA. Mutations in mtDNA can cause fatal or severely debilitating disorders with limited treatment options. Clinical manifestations vary based on mutation type and heteroplasmy (that is, the relative levels of mutant and wild-type mtDNA within each cell). Here we generated genetically corrected pluripotent stem cells (PSCs) from patients with mtDNA disease. Multiple induced pluripotent stem (iPS) cell lines were derived from patients with common heteroplasmic mutations including 3243A>G, causing mitochondrial encephalomyopathy and stroke-like episodes (MELAS), and 8993T>G and 13513G>A, implicated in Leigh syndrome. Isogenic MELAS and Leigh syndrome iPS cell lines were generated containing exclusively wild-type or mutant mtDNA through spontaneous segregation of heteroplasmic mtDNA in proliferating fibroblasts. Furthermore, somatic cell nuclear transfer (SCNT) enabled replacement of mutant mtDNA from homoplasmic 8993T>G fibroblasts to generate corrected Leigh-NT1 PSCs. Although Leigh-NT1 PSCs contained donor oocyte wild-type mtDNA (human haplotype D4a) that differed from Leigh syndrome patient haplotype (F1a) at a total of 47 nucleotide sites, Leigh-NT1 cells displayed transcriptomic profiles similar to those in embryo-derived PSCs carrying wild-type mtDNA, indicative of normal nuclear-to-mitochondrial interactions. Moreover, genetically rescued patient PSCs displayed normal metabolic function compared to impaired oxygen consumption and ATP production observed in mutant cells. We conclude that both reprogramming approaches offer complementary strategies for derivation of PSCs containing exclusively wild-type mtDNA, through spontaneous segregation of heteroplasmic mtDNA in individual iPS cell lines or mitochondrial replacement by SCNT in homoplasmic mtDNA-based disease.


Assuntos
DNA Mitocondrial/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Perfilação da Expressão Gênica , Haplótipos/genética , Humanos , Doença de Leigh/genética , Doença de Leigh/metabolismo , Doença de Leigh/patologia , Camundongos , Mitocôndrias/patologia , Doenças Mitocondriais/patologia , Encefalomiopatias Mitocondriais/genética , Encefalomiopatias Mitocondriais/metabolismo , Encefalomiopatias Mitocondriais/patologia , Mutação/genética , Técnicas de Transferência Nuclear , Nucleotídeos/genética , Consumo de Oxigênio , Polimorfismo de Nucleotídeo Único/genética , Análise de Sequência de RNA , Pele/citologia
11.
Nature ; 511(7508): 177-83, 2014 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-25008523

RESUMO

Human pluripotent stem cells hold potential for regenerative medicine, but available cell types have significant limitations. Although embryonic stem cells (ES cells) from in vitro fertilized embryos (IVF ES cells) represent the 'gold standard', they are allogeneic to patients. Autologous induced pluripotent stem cells (iPS cells) are prone to epigenetic and transcriptional aberrations. To determine whether such abnormalities are intrinsic to somatic cell reprogramming or secondary to the reprogramming method, genetically matched sets of human IVF ES cells, iPS cells and nuclear transfer ES cells (NT ES cells) derived by somatic cell nuclear transfer (SCNT) were subjected to genome-wide analyses. Both NT ES cells and iPS cells derived from the same somatic cells contained comparable numbers of de novo copy number variations. In contrast, DNA methylation and transcriptome profiles of NT ES cells corresponded closely to those of IVF ES cells, whereas iPS cells differed and retained residual DNA methylation patterns typical of parental somatic cells. Thus, human somatic cells can be faithfully reprogrammed to pluripotency by SCNT and are therefore ideal for cell replacement therapies.


Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes/metabolismo , Animais , Linhagem Celular , Aberrações Cromossômicas , Cromossomos Humanos X/genética , Cromossomos Humanos X/metabolismo , Variações do Número de Cópias de DNA , Metilação de DNA , Estudo de Associação Genômica Ampla , Impressão Genômica , Humanos , Técnicas de Transferência Nuclear/normas , Células-Tronco Pluripotentes/citologia , Transcriptoma
12.
J Mol Biol ; 426(3): 645-55, 2014 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-24211469

RESUMO

The catalytic moiety of Pseudomonas exotoxin A (domain III or PE3) inhibits protein synthesis by ADP-ribosylation of eukaryotic elongation factor 2. PE3 is widely used as a cytocidal payload in receptor-targeted protein toxin conjugates. We have designed and characterized catalytically inactive fragments of PE3 that are capable of structural complementation. We dissected PE3 at an extended loop and fused each fragment to one subunit of a heterospecific coiled coil. In vitro ADP-ribosylation and protein translation assays demonstrate that the resulting fusions-supplied exogenously as genetic elements or purified protein fragments-had no significant catalytic activity or effect on protein synthesis individually but, in combination, catalyzed the ADP-ribosylation of eukaryotic elongation factor 2 and inhibited protein synthesis. Although complementing PE3 fragments are catalytically less efficient than intact PE3 in cell-free systems, co-expression in live cells transfected with transgenes encoding the toxin fusions inhibits protein synthesis and causes cell death comparably as intact PE3. Complementation of split PE3 offers a direct extension of the immunotoxin approach to generate bispecific agents that may be useful to target complex phenotypes.


Assuntos
ADP Ribose Transferases/química , ADP Ribose Transferases/metabolismo , Adenosina Difosfato Ribose/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Exotoxinas/química , Exotoxinas/metabolismo , Biossíntese de Proteínas , Fatores de Virulência/química , Fatores de Virulência/metabolismo , Domínio Catalítico , Sobrevivência Celular , Sistema Livre de Células , Cromatografia em Gel , Células HEK293 , Humanos , Immunoblotting , Imunoprecipitação
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