베타액틴-RFP 형질전환 유전자가 이식된 형질전환 바다 송사리 (Oryzias dancena) 계통들의 발현 특징

Abstract

Stable transgenic euryhaline marine medaka (Oryzias dancena) strains carrying red fluorescence protein (RFP) transgene construct (podβ-actRFP) driven by its own cytoskeletal β-actin promoter were characterized with the purpose of selecting the best model transgenic line for environmental risk assessment associated with living genetically modified fishes. Experimental transgenesis with podβ-actRFP in this species showed, in overall, that the regulatory patterns of transgene could be well in accordance with those of endogenous β-actin genes in terms of consistent and ubiquitous expression throughout nearly entire life cycle of the transgenic fish. During embryonic development, the onset of transgene expression was usually detected from the neurula stage and the resultant RFP signals continued to adult at maturity. RFP signals were ubiquitously distributed in all the tissues of transgenic fish examined with various expression levels across tissues. Different transgenic lines showed different transgenic genotypes as judged by Southern blot hybridization analysis and they displayed varying degree of RFP intensities in their external phenotypes. However there was no direct relationship between transgene copy numbers and expression strength, indicating that copy number-dependent expression was not achieved in this transgenic group. All of transgenic founders were mosaic in their germ cells. However, most transgenic individuals belonging to subsequent generations could pass on the fluorescent transgene to their offspring with an expected Mendelian ratio. However, a transgenic line having the highest transgene copy number (>240 copies/cell) showed the unstable pattern of germ-line transmission including the loss of transgene. Interestingly, many transgenic females inherited the RFP proteins to non-transgenic offspring embryos as well as to transgenic offspring. Such non-transgenically transmitted RFP signals was persistent for a considerably long period up to early larval stages, but eventually disappeared with the progress of development and early growth. Results from this study indicate that β-actin promoter is useful for marking whole cells of transgenic fish using fluorescence, which may facilitate the analysis of horizontal and vertical transgene transfer from transgenic fish to other conspecific and/or symbiotic members installed in experimentally designed semi-surrogate ecosystems. However this study suggests also that extensive evaluation of multiple transgenic strains should be made for selecting the suitable and stable transgenic line to be applied to this purpose.