아르테미아(Artemia franciscana)내구란에 외래 유전자 이식을 위한 연구

Abstract

The objective of this study is to observe the result of foreign gene transfer using the cyst of A. franciscana to conduct risk assessment of living genetically modified organisms (LMOs) in the marine ecosystem. The result showed that the optimal time for NaCIO treatment to remove the alveolar layer of A. franciscana cysts was 3.5 min. Supplementary treatment of 20 ppm chloramphenicol and 400 ppm oxytetracycline were found to be most suitable for the axenic culture of A. franciscana. The feasibility of gene integration was analyzed in the part of tissues including antenna, endopodite, telopodite and expedite. In result, PCR analysis was available with a small amount of tissues obtained, however, the Artemia which the tissues were removed did not survive even from the axenic culture conditions. In order to establish the optimal particle bombardment condition for gene transfer on the A. franciscana cysts, 27 variations of experiments were performed on 1.35 g (1 g = approximately 1.8 x 105 cysts) of A. .franciscana cysts. The condition –1.6 um gold particle, 1,350 psi helium pressure, 12 cm target distance and a bombardment – was proven to yield the highest result. Although the integration frequency was 18.4% at the highest, the expression - positive individual was not found. A total of 698 A. franciscana cysts (410 decapsulated) was microinjected and treated with tannic acid to protect the embryonic cuticle layer. Only 10 nauplius were hatched (1.2 ± 2.3% of mean hatchability) but none of them could survive beyond the nauplius stage. Results from this study suggest that the ovoviviparous eggs should be obtained from the mother in order to produce transgenic Artemia through gene transfer trial in the early stage of ontogeny.