RESUMO
Genome editing tools enable efficient and accurate genome manipulation. An enhanced ability to modify the genomes of livestock species could be utilized to improve disease resistance, productivity or breeding capability as well as the generation of new biomedical models. To date, with respect to the direct injection of genome editor mRNA into livestock zygotes, this technology has been limited to the generation of pigs with edited genomes. To capture the far-reaching applications of gene-editing, from disease modelling to agricultural improvement, the technology must be easily applied to a number of species using a variety of approaches. In this study, we demonstrate zygote injection of TALEN mRNA can also produce gene-edited cattle and sheep. In both species we have targeted the myostatin (MSTN) gene. In addition, we report a critical innovation for application of gene-editing to the cattle industry whereby gene-edited calves can be produced with specified genetics by ovum pickup, in vitro fertilization and zygote microinjection (OPU-IVF-ZM). This provides a practical alternative to somatic cell nuclear transfer for gene knockout or introgression of desirable alleles into a target breed/genetic line.
Assuntos
Animais Geneticamente Modificados/genética , Genoma , Miostatina/genética , Carneiro Doméstico/genética , Animais , Cruzamento , Bovinos , Fertilização in vitro , Engenharia Genética , Gado , Técnicas de Transferência Nuclear , ZigotoRESUMO
We describe a fundamentally novel feat of animal genetic engineering: the precise and efficient substitution of an agronomic haplotype into a domesticated species. Zinc finger nuclease in-embryo editing of the RELA locus generated live born domestic pigs with the warthog RELA orthologue, associated with resilience to African Swine Fever. The ability to efficiently achieve interspecies allele introgression in one generation opens unprecedented opportunities for agriculture and basic research.
Assuntos
Resistência à Doença/genética , Edição de Genes/métodos , Engenharia Genética , Ligases/genética , Febre Suína Africana/genética , Febre Suína Africana/virologia , Vírus da Febre Suína Africana/patogenicidade , Alelos , Animais , Genoma , Haplótipos , SuínosRESUMO
Traditional methods of transgene delivery in livestock are inefficient. Recently, human immunodeficiency virus (HIV-1) based lentiviral vectors have been shown to offer an efficient transgene delivery system. We now extend this method by demonstrating efficient generation of transgenic pigs using an equine infectious anaemia virus derived vector. We used this vector to deliver a green fluorescent protein expressing transgene; 31% of injected/transferred eggs resulted in a transgenic founder animal and 95% of founder animals displayed green fluorescence. This compares favourably with results using HIV-1 based vectors, and is substantially more efficient than the standard pronuclear microinjection method, indicating that lentiviral transgene delivery may be a general tool with which to efficiently generate transgenic mammals.
Assuntos
Vírus da Anemia Infecciosa Equina/genética , Proteínas Luminescentes/genética , Animais , Animais Geneticamente Modificados , Southern Blotting , Transferência Embrionária , Feminino , Técnicas de Transferência de Genes , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde , Proteínas Luminescentes/análise , Suínos , ZigotoRESUMO
Kidneys from lambs derived by nuclear transfer are frequently abnormal and are characterized by an enlarged pelvis and narrow medulla, consistent with lower urinary tract obstruction and development of variable hydronephrosis. The precise pathogenesis of this entity is unknown. Immunohistochemical staining for intermediate filaments was used to further characterize the lesions seen in this condition and was compared with age-matched control tissue. Major findings were upregulation of cytokeratin on damaged tubules, desmin and vimentin in undifferentiated mesenchyme, and smooth muscle actin in mesenchyme and on smooth muscle "collars" around dilated tubules. In addition, some cases showed reexpression of vimentin and desmin on proximal tubular epithelial cells. Taken together, these findings provide a valuable database for tracking the expression of intermediate filaments throughout renal development in sheep and have further characterized the nature of the response to injury by the developing kidney, a response that is characterized by proliferation of mesenchyme and both reexpression and upregulation of intermediate filaments within renal cells. In addition, the study has confirmed that the changes in cloned lamb nephropathy are established by day 85 of development.
Assuntos
Clonagem de Organismos/efeitos adversos , Nefropatias/metabolismo , Rim/metabolismo , Actinas/biossíntese , Animais , Desmina/biossíntese , Imuno-Histoquímica , Filamentos Intermediários/metabolismo , Queratinas/biossíntese , Rim/anormalidades , Rim/embriologia , Nefropatias/embriologia , Nefropatias/etiologia , Músculo Liso/metabolismo , Ovinos , Vimentina/biossínteseRESUMO
Transcription activator-like effector nuclease (TALEN) and zinc finger nuclease (ZFN) genome editing technology enables site directed engineering of the genome. Here we demonstrate for the first time that both TALEN and ZFN injected directly into pig zygotes can produce live genome edited pigs. Monoallelic as well as heterozygous and homozygous biallelic events were identified, significantly broadening the use of genome editor technology in livestock by enabling gene knockout in zygotes from any chosen mating.
Assuntos
Animais Geneticamente Modificados/genética , Fertilização in vitro , Engenharia Genética , Genoma , Edição de RNA/genética , Dedos de Zinco/genética , Zigoto/citologia , Alelos , Animais , Animais Geneticamente Modificados/crescimento & desenvolvimento , Sequência de Bases , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Endonucleases/metabolismo , Feminino , Técnicas de Inativação de Genes , Homozigoto , Dados de Sequência Molecular , Técnicas de Transferência Nuclear , Homologia de Sequência do Ácido Nucleico , Suínos , Zigoto/metabolismoAssuntos
Animais Recém-Nascidos/anormalidades , Animais Recém-Nascidos/genética , Clonagem de Organismos/efeitos adversos , Clonagem de Organismos/métodos , Doenças Genéticas Inatas/etiologia , Doenças Genéticas Inatas/genética , Ovinos/anormalidades , Ovinos/genética , Animais , Doenças Genéticas Inatas/patologiaRESUMO
There are continued claims of attempts to clone humans using nuclear transfer, despite the serious problems that have been encountered in cloning other mammals. It is known that epigenetic and genetic mechanisms are involved in clone failure, but we still do not know exactly how. Human reproductive cloning is unethical, but the production of cells from cloned embryos could offer many potential benefits. So, can human cloning be made safe?