Construction of a CRISPR-Cas9 knockdown lentiviral plasmid of goose (Anas platyrhynchos) stearoyl-coenzyme A desaturase gene
In order to further explore the mechanism of endogenous fatty acid synthesis and metabolism in goose granulosa cells, we used CRISPR-Cas9 technology to construct knockdown plasmids of a goose targeted stearoyl-coenzyme A desaturase (SCD) gene and package lentivirus. First, we designed the sequence o...
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| Format: | Article |
| Language: | English |
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Zhejiang University Press
2020-10-01
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| Series: | 浙江大学学报. 农业与生命科学版 |
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| Online Access: | https://www.academax.com/doi/10.3785/j.issn.1008-9209.2020.02.151 |
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| author | YUAN Xin LI Liang HE Hua HU Shenqiang WANG Jiwen |
| author_facet | YUAN Xin LI Liang HE Hua HU Shenqiang WANG Jiwen |
| author_sort | YUAN Xin |
| collection | DOAJ |
| description | In order to further explore the mechanism of endogenous fatty acid synthesis and metabolism in goose granulosa cells, we used CRISPR-Cas9 technology to construct knockdown plasmids of a goose targeted stearoyl-coenzyme A desaturase (SCD) gene and package lentivirus. First, we designed the sequence of single-guide RNA (sgRNA) of goose SCD gene; second, synthesized in vitro and tested the lysis efficacy of sgRNA to target DNA site by endonuclease cleavage assays; finally, prepared the lentiviral plasmid of psgRNA-mCherry-T2A-Puro and pLenti-Cas9-T2A-EGFP using psPAX2 and pMD2.G as package plasmids. Results showed that the lentiviral plasmid was successfully constructed, and the strong double positive cell groups co-expressing the red fluorescent protein (mCherry) and enhanced green fluorescent protein (EGFP) were screened out when the lentiviral plasmid infected the Chinese hamster ovary (CHO) cells. The above results lay the foundation for infecting goose primary granulosa cells and targeting knockdown the SCD gene. |
| format | Article |
| id | doaj-art-363faa17c843449f93f8d18a3a12ba68 |
| institution | DOAJ |
| issn | 1008-9209 2097-5155 |
| language | English |
| publishDate | 2020-10-01 |
| publisher | Zhejiang University Press |
| record_format | Article |
| series | 浙江大学学报. 农业与生命科学版 |
| spelling | doaj-art-363faa17c843449f93f8d18a3a12ba682025-08-20T03:16:05ZengZhejiang University Press浙江大学学报. 农业与生命科学版1008-92092097-51552020-10-014652953810.3785/j.issn.1008-9209.2020.02.15110089209Construction of a CRISPR-Cas9 knockdown lentiviral plasmid of goose (Anas platyrhynchos) stearoyl-coenzyme A desaturase geneYUAN XinLI LiangHE HuaHU ShenqiangWANG JiwenIn order to further explore the mechanism of endogenous fatty acid synthesis and metabolism in goose granulosa cells, we used CRISPR-Cas9 technology to construct knockdown plasmids of a goose targeted stearoyl-coenzyme A desaturase (SCD) gene and package lentivirus. First, we designed the sequence of single-guide RNA (sgRNA) of goose SCD gene; second, synthesized in vitro and tested the lysis efficacy of sgRNA to target DNA site by endonuclease cleavage assays; finally, prepared the lentiviral plasmid of psgRNA-mCherry-T2A-Puro and pLenti-Cas9-T2A-EGFP using psPAX2 and pMD2.G as package plasmids. Results showed that the lentiviral plasmid was successfully constructed, and the strong double positive cell groups co-expressing the red fluorescent protein (mCherry) and enhanced green fluorescent protein (EGFP) were screened out when the lentiviral plasmid infected the Chinese hamster ovary (CHO) cells. The above results lay the foundation for infecting goose primary granulosa cells and targeting knockdown the SCD gene.https://www.academax.com/doi/10.3785/j.issn.1008-9209.2020.02.151stearoyl-coenzyme A desaturase geneCRISPR-Cas9 technologygene site-directed mutagenesisgoose (<italic>Anas platyrhynchos</italic>) |
| spellingShingle | YUAN Xin LI Liang HE Hua HU Shenqiang WANG Jiwen Construction of a CRISPR-Cas9 knockdown lentiviral plasmid of goose (Anas platyrhynchos) stearoyl-coenzyme A desaturase gene 浙江大学学报. 农业与生命科学版 stearoyl-coenzyme A desaturase gene CRISPR-Cas9 technology gene site-directed mutagenesis goose (<italic>Anas platyrhynchos</italic>) |
| title | Construction of a CRISPR-Cas9 knockdown lentiviral plasmid of goose (Anas platyrhynchos) stearoyl-coenzyme A desaturase gene |
| title_full | Construction of a CRISPR-Cas9 knockdown lentiviral plasmid of goose (Anas platyrhynchos) stearoyl-coenzyme A desaturase gene |
| title_fullStr | Construction of a CRISPR-Cas9 knockdown lentiviral plasmid of goose (Anas platyrhynchos) stearoyl-coenzyme A desaturase gene |
| title_full_unstemmed | Construction of a CRISPR-Cas9 knockdown lentiviral plasmid of goose (Anas platyrhynchos) stearoyl-coenzyme A desaturase gene |
| title_short | Construction of a CRISPR-Cas9 knockdown lentiviral plasmid of goose (Anas platyrhynchos) stearoyl-coenzyme A desaturase gene |
| title_sort | construction of a crispr cas9 knockdown lentiviral plasmid of goose anas platyrhynchos stearoyl coenzyme a desaturase gene |
| topic | stearoyl-coenzyme A desaturase gene CRISPR-Cas9 technology gene site-directed mutagenesis goose (<italic>Anas platyrhynchos</italic>) |
| url | https://www.academax.com/doi/10.3785/j.issn.1008-9209.2020.02.151 |
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