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期刊名称: Biology of Reproduction
Volume:91    Issue:3        Page:78-78
ISSN:0006-3363

Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro-derived oocytes and embryos期刊论文

作者: Whitworth Kristin M Lee Kiho Benne Joshua A Beaton Benjamin P Spate Lee D
DOI:10.1095/biolreprod.114.121723

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页码: 78-78
被引频次: 138
出版者: SOC STUDY REPRODUCTION
期刊名称: Biology of Reproduction
ISSN: 0006-3363
卷期: Volume:91    Issue:3
语言: English
摘要: Targeted modification of the pig genome can be challenging. Recent applications of the CRISPR/Cas9 system hold promise for improving the efficacy of genome editing. When a designed CRISPR/Cas9 system targeting CD163 or CD1D was introduced into somatic cells, it was highly efficient in inducing mutations. When these mutated cells were used with somatic cell nuclear transfer, offspring with these modifications were created. When the CRISPR/Cas9 system was delivered into in vitro produced presumptive porcine zygotes, the system was effective in creating mutations in eGFP, CD163, and CD1D (100% targeting efficiency in blastocyst stage embryos); however, it also presented some embryo toxicity. We could also induce deletions in CD163 or CD1D by introducing two types of CRISPRs with Cas9. The system could also disrupt two genes, CD163 and eGFP, simultaneously when two CRISPRs targeting two genes with Cas9 were delivered into zygotes. Direct injection of CRISPR/Cas9 targeting CD163 or CD1D into zygotes resulted in piglets that have mutations on both alleles with only one CD1D pig having a mosaic genotype. We show here that the CRISPR/Cas9 system can be used by two methods. The system can be used to modify somatic cells followed by somatic cell nuclear transfer. System components can also be used in in vitro produced zygotes to generate pigs with specific genetic modifications.
相关主题: Porcine/pig, Embryo, Genetic engineering, CRISPR/Cas9, Somatic cell nuclear transfer, Blastocyst, KNOCKOUT PIGS, embryo, CAS SYSTEM, ZINC-FINGER NUCLEASES, genetic engineering, ONE-STEP GENERATION, somatic cell nuclear transfer, porcine/pig, IN-VITRO, REPRODUCTIVE BIOLOGY, RESPIRATORY SYNDROME VIRUS, PORCINE, GENE-EXPRESSION, blastocyst, CLONING EFFICIENCY, CLONED PIGS, Embryo Culture Techniques - veterinary, Sus scrofa - physiology, Genetic Engineering - veterinary, Nuclear Transfer Techniques - veterinary, Blastocyst - physiology, Male, Green Fluorescent Proteins - genetics, Receptors, Cell Surface - antagonists & inhibitors, Antigens, CD - genetics, Antigens, CD - metabolism, Sus scrofa - genetics, Embryo Transfer - veterinary, Gene Deletion, Antigens, CD1d - genetics, Female, In Vitro Oocyte Maturation Techniques - veterinary, Transgenes, Genetic Engineering - methods, Cell Line, Green Fluorescent Proteins - metabolism, Receptors, Cell Surface - metabolism, Antigens, CD1d - chemistry, Fertilization in Vitro - veterinary, Antigens, CD1d - metabolism, Antigens, Differentiation, Myelomonocytic - genetics, Embryo, Mammalian - physiology, Animals, Oocytes - physiology, Animals, Genetically Modified - genetics, Antigens, Differentiation, Myelomonocytic - metabolism, CRISPR-Cas Systems, Genetic Engineering - adverse effects, Animals, Genetically Modified - physiology, Mutation, Receptors, Cell Surface - genetics, Index Medicus,

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