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1.
Cell ; 168(3): 473-486.e15, 2017 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-28129541

RESUMO

Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.


Assuntos
Quimerismo , Edição de Genes , Mamíferos/embriologia , Animais , Blastocisto , Sistemas CRISPR-Cas , Bovinos , Embrião de Mamíferos/citologia , Feminino , Humanos , Masculino , Mamíferos/classificação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Células-Tronco Pluripotentes , Ratos , Ratos Sprague-Dawley , Sus scrofa
2.
Proc Natl Acad Sci U S A ; 115(9): 2090-2095, 2018 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-29440377

RESUMO

Embryonic stem cells (ESCs) are derived from the inner cell mass of preimplantation blastocysts. From agricultural and biomedical perspectives, the derivation of stable ESCs from domestic ungulates is important for genomic testing and selection, genome engineering, and modeling human diseases. Cattle are one of the most important domestic ungulates that are commonly used for food and bioreactors. To date, however, it remains a challenge to produce stable pluripotent bovine ESC lines. Employing a culture system containing fibroblast growth factor 2 and an inhibitor of the canonical Wnt-signaling pathway, we derived pluripotent bovine ESCs (bESCs) with stable morphology, transcriptome, karyotype, population-doubling time, pluripotency marker gene expression, and epigenetic features. Under this condition bESC lines were efficiently derived (100% in optimal conditions), were established quickly (3-4 wk), and were simple to propagate (by trypsin treatment). When used as donors for nuclear transfer, bESCs produced normal blastocyst rates, thereby opening the possibility for genomic selection, genome editing, and production of cattle with high genetic value.


Assuntos
Blastocisto/fisiologia , Bovinos/embriologia , Células-Tronco Embrionárias/fisiologia , Células-Tronco Pluripotentes/fisiologia , Animais , Biomarcadores , Técnicas de Cultura de Células/veterinária , Diferenciação Celular , Células Cultivadas , Clonagem de Organismos , Técnicas de Cultura Embrionária/veterinária , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Técnicas de Transferência Nuclear/veterinária
3.
Reproduction ; 160(5): 761-772, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33065542

RESUMO

Until recently, it has been difficult to derive and maintain stable embryonic stem cells lines from livestock species. Sheep ESCs with characteristics similar to those described for rodents and primates have not been produced. We report the derivation of sheep ESCs under a chemically defined culture system containing fibroblast growth factor 2 (FGF2) and a tankyrase/Wnt inhibitor (IWR1). We also show that several culture conditions used for stabilizing naïve and intermediate pluripotency states in humans and mice were unsuitable to maintain ovine pluripotency in vitro. Sheep ESCs display a smooth dome-shaped colony morphology, and maintain an euploid karyotype and stable expression of pluripotency markers after more than 40 passages. We further demonstrate that IWR1 and FGF2 are essential for the maintenance of an undifferentiated state in de novo derived sheep ESCs. The derivation of stable pluripotent cell lines from sheep blastocysts represents a step forward toward understanding pluripotency regulation in livestock species and developing novel biomedical and agricultural applications.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Células-Tronco Pluripotentes/citologia , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Células-Tronco Pluripotentes/metabolismo , Ovinos
4.
Transgenic Res ; 27(6): 525-537, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30284144

RESUMO

The production of knock-out (KO) livestock models is both expensive and time consuming due to their long gestational interval and low number of offspring. One alternative to increase efficiency is performing a genetic screening to select pre-implantation embryos that have incorporated the desired mutation. Here we report the use of sheep embryo biopsies for detecting CRISPR/Cas9-induced mutations targeting the gene PDX1 prior to embryo transfer. PDX1 is a critical gene for pancreas development and the target gene required for the creation of pancreatogenesis-disabled sheep. We evaluated the viability of biopsied embryos in vitro and in vivo, and we determined the mutation efficiency using PCR combined with gel electrophoresis and digital droplet PCR (ddPCR). Next, we determined the presence of mosaicism in ~ 50% of the recovered fetuses employing a clonal sequencing methodology. While the use of biopsies did not compromise embryo viability, the presence of mosaicism diminished the diagnostic value of the technique. If mosaicism could be overcome, pre-implantation embryo biopsies for mutation screening represents a powerful approach that will streamline the creation of KO animals.


Assuntos
Animais Geneticamente Modificados , Blastocisto , Sistemas CRISPR-Cas , Embrião de Mamíferos , Edição de Genes/veterinária , Proteínas de Homeodomínio/genética , Mutação , Transativadores/genética , Animais , Biópsia , Transferência Embrionária , Desenvolvimento Embrionário , Feminino , Edição de Genes/métodos , Masculino , Mosaicismo , Ovinos
5.
J Equine Vet Sci ; 89: 103025, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32563448

RESUMO

The breakthrough and rapid advance of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has enabled the efficient generation of gene-edited animals by one-step embryo manipulation. Clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 delivery to the livestock embryos has been typically achieved by intracytoplasmic microinjection; however, recent studies show that electroporation may be a reliable, efficient, and practical method for CRISPR/Cas9 delivery. The source of embryos used to generate gene-edited animals varies from in vivo to in vitro produced, depending mostly on the species of interest. In addition, different Cas9 and gRNA reagents can be used for embryo editing, ranging from Cas9-coding plasmid or messenger RNA to Cas9 recombinant protein, which can be combined with in vitro transcribed or synthetic guide RNAs. Mosaicism is reported as one of the main problems with generation of animals by embryo editing. On the other hand, off-target mutations are rarely found in livestock derived from one-step editing. In this review, we discussed these and other aspects of generating gene-edited animals by single-step embryo manipulation.


Assuntos
Edição de Genes , Gado , Animais , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes/veterinária
7.
Biotechniques ; 65(5): 281-283, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30394131

RESUMO

CRISPR technologies used for mammalian embryology have wide implications from basic research to applications in agriculture and biomedicine. Confirmation of successful gene editing following CRISPR/Cas9 delivery is often limited to either protein expression or sequencing analyses of embryos but not both, due to technical challenges. Herein we report an integrative approach for evaluating both protein expression and genotype of single embryos from fixed bovine embryos previously subjected to CRISPR/Cas9 microinjection. The techniques described facilitate investigation of functional genomics in bovine embryos compatible with gene editing in livestock after zygotic CRISPR microinjection. These methods avoid traditional avenues that necessitate the use of gene-edited cell lines followed by nuclear transfer that hinder efficiency, limit physiological relevance and contribute to technical challenges.


Assuntos
Sistemas CRISPR-Cas , Bovinos/embriologia , Bovinos/genética , Edição de Genes/métodos , Animais , Sequência de Bases , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA/análise , DNA/genética , Embrião de Mamíferos/química , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/ultraestrutura , Imuno-Histoquímica/métodos , Imagem Óptica/métodos , Análise de Sequência de DNA/métodos , Fixação de Tecidos/métodos
8.
Epigenetics ; 12(12): 1048-1056, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29160132

RESUMO

The substantial epigenetic remodeling that occurs during early stages of mammalian embryonic development likely contributes to reprogramming the parental genomes from a differentiated to a totipotent state and activation of the embryonic genome. Trimethylation of lysine 27 of histone 3 (H3K27me3) is a repressive mark that undergoes global dynamic changes during preimplantation development of several species. To ascertain the role of H3K27me3 in bovine preimplantation development we perturbed the activity of KDM6B, which demethylates H3K27me3. Knockdown of maternal KDM6B mRNA inhibited the reduction in global levels of H3K27me3 from 2-cell to 8-cell embryo stages and compromised development to the blastocyst stage; embryos that developed to the blastocyst stage had fewer inner cell mass (ICM) and trophectoderm (TE) cells. In addition, the transcriptome of KDM6B knockdown embryos was altered at the 8-cell stage and characterized by downregulation of transcripts related to transcriptional regulation, chromatin remodeling, and protein catabolism. Inhibiting the catalytic activity of KDM6B with a specific small molecule inhibitor also prevented the global decrease in H3K27me3 and compromised development to the blastocyst stage. These results indicate that histone demethylation activity, mediated by KDM6B, is required for the global decrease in H3K27me3, correct activation of the embryonic genome, and development to the blastocyst stage in bovine embryos.


Assuntos
Blastocisto/metabolismo , Desenvolvimento Embrionário , Código das Histonas , Histona Desmetilases com o Domínio Jumonji/metabolismo , Animais , Bovinos , Células Cultivadas , Feminino , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Histona Desmetilases com o Domínio Jumonji/genética
9.
Sci Rep ; 7(1): 10487, 2017 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-28874671

RESUMO

Genome editing using programmable nucleases has revolutionized biomedical research. CRISPR-Cas9 mediated zygote genome editing enables high efficient production of knockout animals suitable for studying development and relevant human diseases. Here we report efficient disabling pancreatogenesis in pig embryos via zygotic co-delivery of Cas9 mRNA and dual sgRNAs targeting the PDX1 gene, which when combined with chimeric-competent human pluriopotent stem cells may serve as a suitable platform for the xeno-generation of human tissues and organs in pigs.


Assuntos
Sistemas CRISPR-Cas , Terapia Genética/veterinária , Organogênese/genética , Pâncreas/metabolismo , Suínos/genética , Animais , Terapia Genética/métodos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Pâncreas/embriologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/transplante , Transplante de Células-Tronco/métodos , Transplante de Células-Tronco/veterinária , Suínos/embriologia , Coleta de Tecidos e Órgãos/métodos , Coleta de Tecidos e Órgãos/veterinária , Transativadores/genética , Transativadores/metabolismo
10.
Sci Rep ; 7(1): 17472, 2017 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-29234093

RESUMO

One of the ultimate goals of regenerative medicine is the generation of patient-specific organs from pluripotent stem cells (PSCs). Sheep are potential hosts for growing human organs through the technique of blastocyst complementation. We report here the creation of pancreatogenesis-disabled sheep by oocyte microinjection of CRISPR/Cas9 targeting PDX1, a critical gene for pancreas development. We compared the efficiency of target mutations after microinjecting the CRISPR/Cas9 system in metaphase II (MII) oocytes and zygote stage embryos. MII oocyte microinjection reduced lysis, improved blastocyst rate, increased the number of targeted bi-allelic mutations, and resulted in similar degree of mosaicism when compared to zygote microinjection. While the use of a single sgRNA was efficient at inducing mutated fetuses, the lack of complete gene inactivation resulted in animals with an intact pancreas. When using a dual sgRNA system, we achieved complete PDX1 disruption. This PDX1-/- fetus lacked a pancreas and provides the basis for the production of gene-edited sheep as a host for interspecies organ generation. In the future, combining gene editing with CRISPR/Cas9 and PSCs complementation could result in a powerful approach for human organ generation.


Assuntos
Sistemas CRISPR-Cas , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Oócitos/metabolismo , Pâncreas/embriologia , Pâncreas/metabolismo , Transativadores/genética , Transativadores/metabolismo , Animais , Animais Geneticamente Modificados , Cumarínicos , Edição de Genes/métodos , Técnicas de Silenciamento de Genes/métodos , Microinjeções , Pâncreas/patologia , RNA Guia de Cinetoplastídeos/administração & dosagem , Técnicas de Reprodução Assistida , Análise de Sequência de DNA , Ovinos
11.
J Vis Exp ; (116)2016 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-27768068

RESUMO

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can create genetic modifications that will be present in every cell of an adult organism. It can also be used to deliver siRNA, mRNAs or blocking antibodies to study gene function in preimplantation embryos. The conventional technique for microinjecting embryos used in rodents consists of a very thin micropipette that directly penetrates the plasma membrane when advanced into the embryo. When this technique is applied to livestock animals it usually results in low efficiency. This is mainly because in contrast to mice and rats, bovine, ovine, and porcine zygotes have a very dark cytoplasm and a highly elastic plasma membrane that makes visualization during injection and penetration of the plasma membrane hard to achieve. In this protocol, we describe a suitable microinjection method for the delivery of solutions into the cytoplasm of cattle zygotes that has proved to be successful for sheep and pig embryos as well. First, a laser is used to create a hole in the zona pellucida. Then a blunt-end glass micropipette is introduced through the hole and advanced until the tip of the needle reaches about 3/4 into the embryo. Then, the plasma membrane is broken by aspiration of cytoplasmic content inside the needle. Finally, the aspirated cytoplasmic content followed by the solution of interest is injected back into the embryonic cytoplasm. This protocol has been successfully used for the delivery of different solutions into bovine and ovine zygotes with 100% efficiency, minimal lysis, and normal blastocysts development rates.


Assuntos
Gado , Microinjeções/métodos , Zigoto , Animais , Blastocisto , Bovinos , Membrana Celular , Embrião de Mamíferos , Camundongos , Ratos , Ovinos , Suínos , Zona Pelúcida
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