Modification of fatty acid metabolic pathway by transgenesis in nibe croaker (Nibea mitsukurii)

Naoki Kabeya

Marine fishes are generally unable to produce docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) as they are deficient in the key fatty acid-metabolizing enzymes in the DHA/EPA biosynthetic pathway. It is therefore necessary to supplement with fish oil to diets for cultured marine fish species, which is a dietary source of EPA and DHA. However, since fresh water fishes are capable of synthesizing both DHA and EPA, and they presumably express all of the enzymes required for this biosynthetic pathway, we hypothesized that transgenic marine species carrying the aforementioned fatty acid-metabolizing enzymes could be reared without dietary fish oil. As the first step towards this goal, we produced a transgenic marine fish, the nibe croaker (Nibea mitsukurii), carrying a gene encoding a fatty acid-metabolizing enzyme isolated from masu salmon (Oncorhynchus masou). In order to select a promoter that exhibited high activity in the liver, a major organ involved in fatty acid metabolism, abundant transcripts in the liver were identified by EST analysis. The upstream region of the identified gene was isolated by vectorette PCR and then ligated to the masu salmon elongation of very long chain fatty acids protein 2 (elovl2) gene (OmElo2), which has been predicted to catalyze the elongation step required for producing C22 fatty acids from C20 fatty acids. The resulting transgene was then microinjected into fertilized eggs of the nibe croaker and transgenic F1 progeny carrying the OmElo2 gene were produced by crossing the 16 week-old founders with non-transgenic fish. Subsequently, PCR analysis was performed to identify transgenic individuals and to clarify the distribution of OmElo2 transcripts in F1 fish. In addition, the fatty acid compositions of transgenic F2 fish raised with commercial feed were compared to those of non-transgenic fish. The Apo-14 kDa gene was highly expressed in nibe croaker liver. The OmElo2 gene, which was driven by a 3-kb upstream region of Apo-14 kDa, was transferred into the nibe croaker and the resulting founder was raised to maturity. Among the 46 fish that survived to adulthood, 18 mature males were obtained. PCR analysis detected the presence of the OmElo2 gene in the spermatozoa of one of the fish. Using this transgenic founder, we confirmed germline transmission of the transgene to subsequent generations. The OmElo2 gene was highly expressed in the liver of transgenic F1 individuals, and fatty acid analysis revealed that the liver EPA (20:5n-3) content in OmElo2 transgenic F2 individuals decreased (3.3% vs. 7.7%). However, the DPA (22:5n-3) content in transgenic fish was 2.28-fold (4.1% vs. 1.8%) higher than those of nontransgenic individuals. We therefore concluded that transgenesis of fatty acid-metabolic enzymes can be a powerful tool for manipulating fatty acid metabolic pathways in fish.

Modification of fatty acid metabolic pathway by transgenesis in nibe croaker (Nibea mitsukurii)

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