형질전환 바다송사리 Oryzias dancena를 이용한 비생식학적 유전자 전이 연구

Abstract

Genetically modified organisms (GMOs) or living modified organisms (LMOs) are continuously increased every year in the world. However, concerns have been raised over the potential risks that LMOs pose to human health and the environment. In particular, LM (living modified) fishes are more likely to be a threat than GM (genetically modified) crops to an entire ecosystem because they are commonly transferred alive. Firstly, the objective of this study is to develop the model fish by using the euryhaline marine medaka (Oryzias dancena) to conduct risk assessment of LMOs on the fisheries environments and marine ecosystems. The model fish showed strong expression of red fluorescence protein (RFP) at fast skeletal muscle throughout its life cycle. After microinjection of the podmlc2RFP, the mean mean hatching rate of eggs was 34.8 ± 11.4%. At sexual maturity, 5 individuals (founders) showed RFP signals in their external body. Among 5 founders tested, 2 founders were shown to be able to pass on the fluorescent transgene to their F1 offspring. The frequency of germ-line transmission in the ML-2, 8.8 ± 4.1% was higher than in the ML-1, 5.8 ± 5.2%. During embryonic development, the onset of RFP signals was first detectable at stage 25 (onset of retinal pigmentation stage). The intensity of the RFP signals was gradually increased. At the pre-hatching stage, the RFP positive embryos showed a strong RFP signals at muscle. In adult ML-2 line, an brilliant RFP signals were over the fast skeletal muscle. The intensity of RFP signals were consistently stable in the fish. Secondly, the objective of this study is to determine the risk of transgene transfer by predation in non-reproductive pathway for 24 days. The DNA solutions were mixed to a powder of commercial diet containing structure gene of GM events. After the 24-day feeding trial, tissue samples were collected for analysis of PCR. All the three primer pairs sequentially amplifying the same target genes (PAT gene, DsRed2 gene) could successfully amplify the DNA sequence of interest, but show differences depending on the amplification specificity and sensitivity. And the target genes were detected in all samples after 24 days of feeding, however, after fasting for 5 days, the target genes were not detected in any more all samples. The fish diet containing crystalline and herbicide-tolerant genes in several GM maizes (MON810, GA21, NK603, T25, TC1507 and Bt11) as detected by PCR was fed to fish and it determined whether DNA fragments derived from fish diet could be transferred to O. dancena. The herbicide-tolerant GM events (GA21, NK603 and T25) were detected higher than other GM events. And after fasting for 8 days, the GM events were not detected in all samples, indicating that trans-protein survived longer than transgene. Thirdly, the objective of this study is to focus on the fate of foreign DNA fragments to investigate their survival through the fish gastrointestinal (GI) tract and blood, whether transgenic foreign DNA fragment could be traced in a variety of fish tissues by PCR analysis using O. dancena. So, O. dancena were fed by one of the four experimental diets containing GM maize (MON89034) for 30 weeks. This compared GM maize with commercial maize in feeding O. dancena and investigated the effects of fish feeds including target GM maize on growth performance, egg development and histological analysis of various tissues from the fish. It also compared the experimental diet with Artemia nauplii in feeding O. dancena, and examined differences in the transgene of GM maize and endogenous gene of corn or A. nauplii. No significant differences of results were found among fish fed either the GM maize or A. nauplii. And endogenous genes of maize were also found in a variety of tissues of O. dancena. However, their frequencies were higher than that of the transgene. No significant effect of GM maize on survival of O. dancena were observed.