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Myostatin gene editing by CRISPR/Cas9 technology of Brangus fetal fibroblasts to produce edited embryos by cloning

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dc.contributor.author Bastón, Juan Ignacio
dc.contributor.author Viale, Diego Luis
dc.contributor.author Olguín, Martín
dc.contributor.author Wiedenmann, Elisabet
dc.contributor.author Arnold, Victoria
dc.contributor.author Suva, Mariana
dc.contributor.author Luzzani, Carlos Daniel
dc.contributor.author Miriuka, Santiago Gabriel
dc.contributor.author Moro, Lucía Natalia
dc.contributor.author Vichera, Gabriel
dc.date.accessioned 2021-01-13T16:02:04Z
dc.date.available 2021-01-13T16:02:04Z
dc.date.issued 2021-01-08
dc.identifier.citation Bastón J. I., Viale D., Olguin M., Wiedenmann E., Arnold V., Suva M., Luzzani C.D., Miriuka S., Moro L. N., Vichera G. Myostatin gene editing by CRISPR/Cas9 technology of Brangus fetal fibroblasts to produce edited embryos by cloning. Reproduction, Fertility and Development. 2021; 33, 154-154. doi: 10.1071/RDv33n2Ab94 en_US
dc.identifier.uri https://doi.org/10.1071/RDv33n2Ab94
dc.identifier.uri https://repositorio.fleni.org.ar/handle/123456789/308
dc.description.abstract Some European cattle breeds, such as Charolais and Maine Anjou, have natural mutations in the myostatin gene (MSTN) that inhibit its expression and result in an increase in muscle mass and protein content. An innovative hallmark would be the in vitro introduction of this genotype in South America cattle breeds to improve their commercial value. To achieve this, we aimed to disrupt MSTN gene expression in bovine fetal fibroblasts (BFFs) using CRISPR/Cas9 technology and to generate MSTN-edited embryos by somatic cell nuclear transfer (SCNT). BFFs were isolated from a cloned fetus of a Brangus bull with a prized genetic background and nucleofected with the Cas9 ribonucleoprotein-gRNA complex previously assessed to target exon 2 of the bovine MSTN gene. To evaluate MSTN editing, genomic DNA from the wild-type (WT) and nucleofected BFFs were isolated, exon 2 of MSTN was amplified by PCR, and the PCR product was Sanger sequenced. In all cases, the sequencing results were analysed using the indel Synthego software tool. According to indel analysis, MSTN gene editing efficiency of the BFFs was 58.83% ± 3.2 (n = 6). The resulting edit consisted of insertion of thymine in exon 2 of the MSTN gene that shifted the gene reading frame, introducing a premature stop codon and generating a truncated MSTN protein. The nucleofected BFFs were then used to generate embryos by SCNT, and WT BFFs were included as controls. Embryo cleavage and blastocyst development rates were evaluated at Day 2 and 7, respectively (Chi-squared test, P < 0.05). Although lower cleavage rates were obtained in the MSTN-edited BFFs group [65.3% (n = 273/418) vs. 87.6% (n = 169/193)], no differences were observed at the blastocyst stage [19.1% (n = 80/418) vs. 25.4% (n = 49/193)]. To confirm the efficiency of MSTN editing in the cloned embryos, 10 blastocysts generated with the MSTN-edited BFFs were individually analysed for MSTN exon 2 sequence as described before. The results showed that 30% of the blastocysts (3/10) presented a homozygous biallelic edition, which consisted of a thymine base insertion, as expected. In summary, the strategy we used allowed production of MSTN null cloned Brangus embryos avoiding putative undesired integration of exogenous DNA into the bovine genome, such as plasmid sequence, regardless of off-target occurrences. We conclude that CRISPR/Cas9 is an efficient technique to disrupts MSTN gene expression in bovine embryos; this work represents a step toward improving the production efficiency of South American cattle breeds. en_US
dc.language.iso eng en_US
dc.publisher Csiro Publishing en_US
dc.rights info:eu-repo/semantics/openAccess
dc.rights.uri https://creativecommons.org/licenses/by/2.5/ar/
dc.subject Myostatin en_US
dc.subject Miostatina en_US
dc.subject CRISPR-Cas Systems en_US
dc.subject Sistemas CRISPR-Cas en_US
dc.title Myostatin gene editing by CRISPR/Cas9 technology of Brangus fetal fibroblasts to produce edited embryos by cloning en_US
dc.type info:eu-repo/semantics/publishedVersion
dc.type info:eu-repo/semantics/other en_US
dc.description.fil Fil: Bastón, Juan Ignacio. Kheiron Biotech; Argentina.
dc.description.fil Fil: Viale, Diego Luis. Universidad Nacional de San Martín; Argentina.
dc.description.fil Fil: Olguin, Martín. Kheiron Biotech; Argentina.
dc.description.fil Fil: Wiedenmann, Elisabet. Kheiron Biotech; Argentina.
dc.description.fil Fil: Arnold, Victoria. Kheiron Biotech; Argentina.
dc.description.fil Fil: Suva, Mariana. Kheiron Biotech; Argentina.
dc.description.fil Fil: Luzzani, Carlos Daniel. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.
dc.description.fil Fil: Miriuka, Santiago Gabriel. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.
dc.description.fil Fil: Moro, Lucía Natalia. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.
dc.description.fil Fil: Vichera, Gabriel. Kheiron Biotech; Argentina.
dc.relation.ispartofVOLUME 33
dc.relation.ispartofNUMBER 2
dc.relation.ispartofPAGINATION 154
dc.relation.ispartofCOUNTRY Australia
dc.relation.ispartofCITY Clayton
dc.relation.ispartofTITLE Reproduction, Fertility and Development
dc.relation.ispartofISSN 1448-5990
dc.type.snrd info:ar-repo/semantics/artículo es_ES


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