Production of MSTN Gene-Edited Embryos of Buffalo Using the CRISPR/Cas9 System and SCNT.
Cell Reprogram
; 25(3): 121-127, 2023 06.
Article
em En
| MEDLINE
| ID: mdl-37042654
ABSTRACT
The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system and somatic cell nuclear transfer (SCNT) have been used to produce genome-edited farm animal species for improved production and health traits; however, these tools are rarely used in the buffalo and can play a pivotal role in milk and meat production in tropical and subtropical countries. In this study, we aimed to produce myostatin (MSTN) gene-edited embryos of the Murrah buffalo using the CRISPR/Cas9 system and SCNT. For this, fibroblast cells were electroporated with sgRNAs carrying all-in-one CRISPR/Cas9 plasmids targeting the first exon of the MSTN gene. Following puromycin selection, single-cell clonal populations were established and screened using the TA cloning and Sanger sequencing methods. Of eight single-cell clonal populations, one with a monoallelic and another with a biallelic heterozygous gene editing event were identified. These two gene-edited clonal cell populations were successfully used to produce blastocyst-stage embryos using the handmade cloning method. This work establishes the technical foundation for generation of genome-edited cloned embryos in the buffalo.
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Base de dados:
MEDLINE
Assunto principal:
Sistemas CRISPR-Cas
/
Edição de Genes
Idioma:
En
Ano de publicação:
2023
Tipo de documento:
Article