Construction of Gene Expression Vector and Its Catalysis Efficiency in Bovine Fetal Fibroblast Cells
The FAT-1 protein is an n-3 fatty acid desaturase, which can recognize a range of 18- and 20-carbon n-6 substrates and transform n-6 polyunsaturated fatty acids (PUFAs) into n-3 PUFAs while n-3 PUFAs have beneficial effect on human health. Fat1 gene is the coding sequence from Caenorhabditis elegans...
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| Format: | Article |
| Language: | English |
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Asian-Australasian Association of Animal Production Societies
2012-05-01
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| Series: | Asian-Australasian Journal of Animal Sciences |
| Subjects: | |
| Online Access: | http://www.ajas.info/upload/pdf/25-79.pdf |
| _version_ | 1818683910327894016 |
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| author | Boyang Liu Runjun Yang Junya Li Lupei Zhang Jing Liu Chunyan Lu Chuanjiang Lian Zezhong Li Yonghong Zhang Liying Zhang Zhihui Zhao |
| author_facet | Boyang Liu Runjun Yang Junya Li Lupei Zhang Jing Liu Chunyan Lu Chuanjiang Lian Zezhong Li Yonghong Zhang Liying Zhang Zhihui Zhao |
| author_sort | Boyang Liu |
| collection | DOAJ |
| description | The FAT-1 protein is an n-3 fatty acid desaturase, which can recognize a range of 18- and 20-carbon n-6 substrates and transform n-6 polyunsaturated fatty acids (PUFAs) into n-3 PUFAs while n-3 PUFAs have beneficial effect on human health. Fat1 gene is the coding sequence from Caenorhabditis elegans which might play an important role on lipometabolism. To reveal the function of fat1 gene in bovine fetal fibroblast cells and gain the best cell nuclear donor for transgenic bovines, the codon of fat1 sequence was optimized based on the codon usage frequency preference of bovine muscle protein, and directionally cloned into the eukaryotic expression vector pEF-GFP. After identifying by restrictive enzyme digests with AatII/XbaI and sequencing, the fusion plasmid pEF-GFP-fat1 was identified successfully. The pEF-GFP-fat1 vector was transfected into bovine fetal fibroblast cells mediated by Lipofectamine2000TM. The positive bovine fetal fibroblast cells were selected by G418 and detected by RT-PCR. The results showed that a 1,234 bp transcription was amplified by reverse transcription PCR and the positive transgenic fat1 cell line was successfully established. Then the expression level of fat1 gene in positive cells was detected using quantitative PCR, and the catalysis efficiency was detected by gas chromatography. The results demonstrated that the catalysis efficiency of fat1 was significantly high, which can improve the total PUFAs rich in EPA, DHA and DPA. Construction and expression of pEF-GFP-fat1 vector should be helpful for further understanding the mechanism of regulation of fat1 in vitro. It could also be the first step in the production of fat1 transgenic cattle. |
| first_indexed | 2024-12-17T10:42:14Z |
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| issn | 1011-2367 1976-5517 |
| language | English |
| last_indexed | 2024-12-17T10:42:14Z |
| publishDate | 2012-05-01 |
| publisher | Asian-Australasian Association of Animal Production Societies |
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| series | Asian-Australasian Journal of Animal Sciences |
| spelling | doaj.art-861bfcfb1bfa4de9aa9fb5ac706ffcba2022-12-21T21:52:13ZengAsian-Australasian Association of Animal Production SocietiesAsian-Australasian Journal of Animal Sciences1011-23671976-55172012-05-0125562162810.5713/ajas.2011.1149522722Construction of Gene Expression Vector and Its Catalysis Efficiency in Bovine Fetal Fibroblast CellsBoyang Liu0Runjun Yang1Junya Li2Lupei Zhang3Jing Liu4Chunyan Lu5Chuanjiang Lian6Zezhong Li7Yonghong Zhang8Liying Zhang9Zhihui Zhao10 College of Animal Science and Veterinary Medicine, and Jilin Provincial Key Laboratory of Animal Embryo Engineering, Jilin University, 5333 Xi’an Road, Changchun, Jilin Province, 130062, China College of Animal Science and Veterinary Medicine, and Jilin Provincial Key Laboratory of Animal Embryo Engineering, Jilin University, 5333 Xi’an Road, Changchun, Jilin Province, 130062, China Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Yuanmingyuan West Road No.2, Beijing, 100193, .China Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Yuanmingyuan West Road No.2, Beijing, 100193, .China College of Animal Science and Veterinary Medicine, and Jilin Provincial Key Laboratory of Animal Embryo Engineering, Jilin University, 5333 Xi’an Road, Changchun, Jilin Province, 130062, China College of Animal Science and Veterinary Medicine, and Jilin Provincial Key Laboratory of Animal Embryo Engineering, Jilin University, 5333 Xi’an Road, Changchun, Jilin Province, 130062, China College of Animal Science and Veterinary Medicine, and Jilin Provincial Key Laboratory of Animal Embryo Engineering, Jilin University, 5333 Xi’an Road, Changchun, Jilin Province, 130062, China College of Animal Science and Veterinary Medicine, and Jilin Provincial Key Laboratory of Animal Embryo Engineering, Jilin University, 5333 Xi’an Road, Changchun, Jilin Province, 130062, China College of Animal Science and Veterinary Medicine, and Jilin Provincial Key Laboratory of Animal Embryo Engineering, Jilin University, 5333 Xi’an Road, Changchun, Jilin Province, 130062, China College of Animal Science and Veterinary Medicine, and Jilin Provincial Key Laboratory of Animal Embryo Engineering, Jilin University, 5333 Xi’an Road, Changchun, Jilin Province, 130062, China College of Animal Science and Veterinary Medicine, and Jilin Provincial Key Laboratory of Animal Embryo Engineering, Jilin University, 5333 Xi’an Road, Changchun, Jilin Province, 130062, ChinaThe FAT-1 protein is an n-3 fatty acid desaturase, which can recognize a range of 18- and 20-carbon n-6 substrates and transform n-6 polyunsaturated fatty acids (PUFAs) into n-3 PUFAs while n-3 PUFAs have beneficial effect on human health. Fat1 gene is the coding sequence from Caenorhabditis elegans which might play an important role on lipometabolism. To reveal the function of fat1 gene in bovine fetal fibroblast cells and gain the best cell nuclear donor for transgenic bovines, the codon of fat1 sequence was optimized based on the codon usage frequency preference of bovine muscle protein, and directionally cloned into the eukaryotic expression vector pEF-GFP. After identifying by restrictive enzyme digests with AatII/XbaI and sequencing, the fusion plasmid pEF-GFP-fat1 was identified successfully. The pEF-GFP-fat1 vector was transfected into bovine fetal fibroblast cells mediated by Lipofectamine2000TM. The positive bovine fetal fibroblast cells were selected by G418 and detected by RT-PCR. The results showed that a 1,234 bp transcription was amplified by reverse transcription PCR and the positive transgenic fat1 cell line was successfully established. Then the expression level of fat1 gene in positive cells was detected using quantitative PCR, and the catalysis efficiency was detected by gas chromatography. The results demonstrated that the catalysis efficiency of fat1 was significantly high, which can improve the total PUFAs rich in EPA, DHA and DPA. Construction and expression of pEF-GFP-fat1 vector should be helpful for further understanding the mechanism of regulation of fat1 in vitro. It could also be the first step in the production of fat1 transgenic cattle.http://www.ajas.info/upload/pdf/25-79.pdfGene ExpressionCatalysis EfficiencyBovine Fetal Fibroblast Cells |
| spellingShingle | Boyang Liu Runjun Yang Junya Li Lupei Zhang Jing Liu Chunyan Lu Chuanjiang Lian Zezhong Li Yonghong Zhang Liying Zhang Zhihui Zhao Construction of Gene Expression Vector and Its Catalysis Efficiency in Bovine Fetal Fibroblast Cells Asian-Australasian Journal of Animal Sciences Gene Expression Catalysis Efficiency Bovine Fetal Fibroblast Cells |
| title | Construction of Gene Expression Vector and Its Catalysis Efficiency in Bovine Fetal Fibroblast Cells |
| title_full | Construction of Gene Expression Vector and Its Catalysis Efficiency in Bovine Fetal Fibroblast Cells |
| title_fullStr | Construction of Gene Expression Vector and Its Catalysis Efficiency in Bovine Fetal Fibroblast Cells |
| title_full_unstemmed | Construction of Gene Expression Vector and Its Catalysis Efficiency in Bovine Fetal Fibroblast Cells |
| title_short | Construction of Gene Expression Vector and Its Catalysis Efficiency in Bovine Fetal Fibroblast Cells |
| title_sort | construction of gene expression vector and its catalysis efficiency in bovine fetal fibroblast cells |
| topic | Gene Expression Catalysis Efficiency Bovine Fetal Fibroblast Cells |
| url | http://www.ajas.info/upload/pdf/25-79.pdf |
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