Lipofection-Mediated Introduction of CRISPR/Cas9 System into Porcine Oocytes and Embryos
Liposome-mediated gene transfer has become an alternative method for establishing a gene targeting framework, and the production of mutant animals may be feasible even in laboratories without specialized equipment. However, how this system functions in mammalian oocytes and embryos remains unclear....
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-02-01
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| Series: | Animals |
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| Online Access: | https://www.mdpi.com/2076-2615/11/2/578 |
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| author | Maki Hirata Manita Wittayarat Zhao Namula Quynh Anh Le Qingyi Lin Koki Takebayashi Chommanart Thongkittidilok Fuminori Tanihara Takeshige Otoi |
| author_facet | Maki Hirata Manita Wittayarat Zhao Namula Quynh Anh Le Qingyi Lin Koki Takebayashi Chommanart Thongkittidilok Fuminori Tanihara Takeshige Otoi |
| author_sort | Maki Hirata |
| collection | DOAJ |
| description | Liposome-mediated gene transfer has become an alternative method for establishing a gene targeting framework, and the production of mutant animals may be feasible even in laboratories without specialized equipment. However, how this system functions in mammalian oocytes and embryos remains unclear. The present study was conducted to clarify whether blastocyst genome editing can be performed by treatment with lipofection reagent, guide RNA, and Cas9 for 5 h without using electroporation or microinjection. A mosaic mutation was observed in blastocysts derived from zona pellucida (ZP)-free oocytes following lipofection treatment, regardless of the target genes. When lipofection treatment was performed after in vitro fertilization (IVF), no significant differences in the mutation rates or mutation efficiency were found between blastocysts derived from embryos treated at 24 and 29 h from the start of IVF. Only blastocysts from embryos exposed to lipofection treatment at 29 h after IVF contained biallelic mutant. Furthermore, there were no significant differences in the mutation rates or mutation efficiency between blastocysts derived from embryos at the 2- and 4-cell stages. This suggests that lipofection-mediated gene editing can be performed in ZP-free oocytes and ZP-free embryos; however, other factors affecting the system efficiency should be further investigated. |
| first_indexed | 2024-03-09T00:37:20Z |
| format | Article |
| id | doaj.art-46872bfce2ae42f59b4fcad07a506d1f |
| institution | Directory Open Access Journal |
| issn | 2076-2615 |
| language | English |
| last_indexed | 2024-03-09T00:37:20Z |
| publishDate | 2021-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Animals |
| spelling | doaj.art-46872bfce2ae42f59b4fcad07a506d1f2023-12-11T18:03:09ZengMDPI AGAnimals2076-26152021-02-0111257810.3390/ani11020578Lipofection-Mediated Introduction of CRISPR/Cas9 System into Porcine Oocytes and EmbryosMaki Hirata0Manita Wittayarat1Zhao Namula2Quynh Anh Le3Qingyi Lin4Koki Takebayashi5Chommanart Thongkittidilok6Fuminori Tanihara7Takeshige Otoi8Faculty of Bioscience and Bioindustry, Tokushima University, Tokushima 770-8501, JapanFaculty of Veterinary Science, Prince of Songkla University, Songkhla 90110, ThailandFaculty of Bioscience and Bioindustry, Tokushima University, Tokushima 770-8501, JapanFaculty of Bioscience and Bioindustry, Tokushima University, Tokushima 770-8501, JapanFaculty of Bioscience and Bioindustry, Tokushima University, Tokushima 770-8501, JapanFaculty of Bioscience and Bioindustry, Tokushima University, Tokushima 770-8501, JapanAkkhraratchakumari Veterinary College, Walailak University, Nakorn Sri Thammarat 80161, ThailandFaculty of Bioscience and Bioindustry, Tokushima University, Tokushima 770-8501, JapanFaculty of Bioscience and Bioindustry, Tokushima University, Tokushima 770-8501, JapanLiposome-mediated gene transfer has become an alternative method for establishing a gene targeting framework, and the production of mutant animals may be feasible even in laboratories without specialized equipment. However, how this system functions in mammalian oocytes and embryos remains unclear. The present study was conducted to clarify whether blastocyst genome editing can be performed by treatment with lipofection reagent, guide RNA, and Cas9 for 5 h without using electroporation or microinjection. A mosaic mutation was observed in blastocysts derived from zona pellucida (ZP)-free oocytes following lipofection treatment, regardless of the target genes. When lipofection treatment was performed after in vitro fertilization (IVF), no significant differences in the mutation rates or mutation efficiency were found between blastocysts derived from embryos treated at 24 and 29 h from the start of IVF. Only blastocysts from embryos exposed to lipofection treatment at 29 h after IVF contained biallelic mutant. Furthermore, there were no significant differences in the mutation rates or mutation efficiency between blastocysts derived from embryos at the 2- and 4-cell stages. This suggests that lipofection-mediated gene editing can be performed in ZP-free oocytes and ZP-free embryos; however, other factors affecting the system efficiency should be further investigated.https://www.mdpi.com/2076-2615/11/2/578CRISPR/Cas9embryolipofectionoocytepig |
| spellingShingle | Maki Hirata Manita Wittayarat Zhao Namula Quynh Anh Le Qingyi Lin Koki Takebayashi Chommanart Thongkittidilok Fuminori Tanihara Takeshige Otoi Lipofection-Mediated Introduction of CRISPR/Cas9 System into Porcine Oocytes and Embryos Animals CRISPR/Cas9 embryo lipofection oocyte pig |
| title | Lipofection-Mediated Introduction of CRISPR/Cas9 System into Porcine Oocytes and Embryos |
| title_full | Lipofection-Mediated Introduction of CRISPR/Cas9 System into Porcine Oocytes and Embryos |
| title_fullStr | Lipofection-Mediated Introduction of CRISPR/Cas9 System into Porcine Oocytes and Embryos |
| title_full_unstemmed | Lipofection-Mediated Introduction of CRISPR/Cas9 System into Porcine Oocytes and Embryos |
| title_short | Lipofection-Mediated Introduction of CRISPR/Cas9 System into Porcine Oocytes and Embryos |
| title_sort | lipofection mediated introduction of crispr cas9 system into porcine oocytes and embryos |
| topic | CRISPR/Cas9 embryo lipofection oocyte pig |
| url | https://www.mdpi.com/2076-2615/11/2/578 |
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