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Enhanced production of docosahexaenoic acid in mammalian cells.

Zhu G, Jiang X, Ou Q, Zhang T, Wang M, Sun G, Wang Z, Sun J, Ge T - PLoS ONE (2014)

Bottom Line: By using transient transfection method, Siganus canaliculatus Δ4 desaturase was heterologously expressed in chinese hamster ovary (CHO) cells, and simultaneously, mouse Δ6-desaturase and Δ5-desaturase were overexpressed.The results demonstrated that the overexpression of Δ6/Δ5-desaturases significantly enhanced the ability of transfected cells to convert the added ALA to docosapentaenoic acid (DPA) which in turn get converted into DHA directly and efficiently by the heterologously expressed Δ4 desaturase.This technology provides the basis for potential utility of these gene constructs in the creation of transgenic livestock for increased production of DHA/related products to meet the growing demand of this important PUFA.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry and Molecular Biology, College of Basic Medicine, Jiamusi University, Jiamusi, Heilongjiang, China.

ABSTRACT
Docosahexaenoic acid (DHA), one of the important polyunsaturated fatty acids (PUFA) with pharmaceutical and nutraceutical effects, may be obtained through diet or synthesized in vivo from dietary a-linolenic acid (ALA). However, the accumulation of DHA in human body or other mammals relies on the intake of high dose of DHA for a certain period of time, and the bioconversion of dietary ALA to DHA is very limited. Therefore the mammalian cells are not rich in DHA. Here, we report a new technology for increased production of DHA in mammalian cells. By using transient transfection method, Siganus canaliculatus Δ4 desaturase was heterologously expressed in chinese hamster ovary (CHO) cells, and simultaneously, mouse Δ6-desaturase and Δ5-desaturase were overexpressed. The results demonstrated that the overexpression of Δ6/Δ5-desaturases significantly enhanced the ability of transfected cells to convert the added ALA to docosapentaenoic acid (DPA) which in turn get converted into DHA directly and efficiently by the heterologously expressed Δ4 desaturase. This technology provides the basis for potential utility of these gene constructs in the creation of transgenic livestock for increased production of DHA/related products to meet the growing demand of this important PUFA.

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Partial gas chromatograph traces showing fatty acid profiles of total cellular lipids extracted from the control cells transfected with pcDNA3.1-EGFP (A), and the cells infected with pcDNA3.1-sScD4 (B).
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pone-0096503-g002: Partial gas chromatograph traces showing fatty acid profiles of total cellular lipids extracted from the control cells transfected with pcDNA3.1-EGFP (A), and the cells infected with pcDNA3.1-sScD4 (B).

Mentions: To test whether pcDNA3.1-sScD4 could be expressed in mammalian cells, the vector was introduced into CHO cells, by using liposome transfection method. DPA was added to the cell culture medium for 3 days. The RT-PCR results showed mRNA of this synthetic gene could be greatly transcribed, while the cells transfected with the control plasmid pcDNA3.1-EGFP, which carries the enhanced green fluorescent protein as reporter gene, detected no sScD4 mRNA (Fig.1). The transient transfections of pcDNA3.1-sScD4 in CHO cells confirmed sScD4 can directly and efficiently convert DPA to DHA, as shown in Fig 2. Table 1 shows fatty acids composition of total cellular lipids from the CHO cells transfected with EGFP or sScD4. None of the percentage distribution of a fatty acid varied between the CHO cells transfected with EGFP and sScD4 except DPA and DHA. The percentage distribution of DPA decreased from 20.2±1.62 in CHO cells transfected with EGFP to 14.4±1.46 in the cells transfected with sScD4, while the percentage distribution of DHA increased from 4.0±0.95 in cells transfected with EGFP to 9.2±0.94 in the cells transfected with sScD4.


Enhanced production of docosahexaenoic acid in mammalian cells.

Zhu G, Jiang X, Ou Q, Zhang T, Wang M, Sun G, Wang Z, Sun J, Ge T - PLoS ONE (2014)

Partial gas chromatograph traces showing fatty acid profiles of total cellular lipids extracted from the control cells transfected with pcDNA3.1-EGFP (A), and the cells infected with pcDNA3.1-sScD4 (B).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4008533&req=5

pone-0096503-g002: Partial gas chromatograph traces showing fatty acid profiles of total cellular lipids extracted from the control cells transfected with pcDNA3.1-EGFP (A), and the cells infected with pcDNA3.1-sScD4 (B).
Mentions: To test whether pcDNA3.1-sScD4 could be expressed in mammalian cells, the vector was introduced into CHO cells, by using liposome transfection method. DPA was added to the cell culture medium for 3 days. The RT-PCR results showed mRNA of this synthetic gene could be greatly transcribed, while the cells transfected with the control plasmid pcDNA3.1-EGFP, which carries the enhanced green fluorescent protein as reporter gene, detected no sScD4 mRNA (Fig.1). The transient transfections of pcDNA3.1-sScD4 in CHO cells confirmed sScD4 can directly and efficiently convert DPA to DHA, as shown in Fig 2. Table 1 shows fatty acids composition of total cellular lipids from the CHO cells transfected with EGFP or sScD4. None of the percentage distribution of a fatty acid varied between the CHO cells transfected with EGFP and sScD4 except DPA and DHA. The percentage distribution of DPA decreased from 20.2±1.62 in CHO cells transfected with EGFP to 14.4±1.46 in the cells transfected with sScD4, while the percentage distribution of DHA increased from 4.0±0.95 in cells transfected with EGFP to 9.2±0.94 in the cells transfected with sScD4.

Bottom Line: By using transient transfection method, Siganus canaliculatus Δ4 desaturase was heterologously expressed in chinese hamster ovary (CHO) cells, and simultaneously, mouse Δ6-desaturase and Δ5-desaturase were overexpressed.The results demonstrated that the overexpression of Δ6/Δ5-desaturases significantly enhanced the ability of transfected cells to convert the added ALA to docosapentaenoic acid (DPA) which in turn get converted into DHA directly and efficiently by the heterologously expressed Δ4 desaturase.This technology provides the basis for potential utility of these gene constructs in the creation of transgenic livestock for increased production of DHA/related products to meet the growing demand of this important PUFA.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry and Molecular Biology, College of Basic Medicine, Jiamusi University, Jiamusi, Heilongjiang, China.

ABSTRACT
Docosahexaenoic acid (DHA), one of the important polyunsaturated fatty acids (PUFA) with pharmaceutical and nutraceutical effects, may be obtained through diet or synthesized in vivo from dietary a-linolenic acid (ALA). However, the accumulation of DHA in human body or other mammals relies on the intake of high dose of DHA for a certain period of time, and the bioconversion of dietary ALA to DHA is very limited. Therefore the mammalian cells are not rich in DHA. Here, we report a new technology for increased production of DHA in mammalian cells. By using transient transfection method, Siganus canaliculatus Δ4 desaturase was heterologously expressed in chinese hamster ovary (CHO) cells, and simultaneously, mouse Δ6-desaturase and Δ5-desaturase were overexpressed. The results demonstrated that the overexpression of Δ6/Δ5-desaturases significantly enhanced the ability of transfected cells to convert the added ALA to docosapentaenoic acid (DPA) which in turn get converted into DHA directly and efficiently by the heterologously expressed Δ4 desaturase. This technology provides the basis for potential utility of these gene constructs in the creation of transgenic livestock for increased production of DHA/related products to meet the growing demand of this important PUFA.

Show MeSH