Inducible expression of green fluorescent protein within channel catfish cells by a cecropin gene promoter

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The activity of an insect promoter of the cecropin B gene (Cec B) was investigated using green fluorescent protein (gfp) as a reporter in cells of channel catfish (Ictalurus punctatus). The expression vector pQZ-1 containing the Cec B promoter and a modified gfp cDNA sequence was delivered by lipofection to three catfish cell types: fibroblast and leukocyte cell lines, and primary cultures of leukocytes. No resistance genes were included in the vector for selection of GFP-expressing cells. The GFP mRNA was detected in all three cell types with 5 to 10 times higher concentrations observed in leukocytes than in fibroblasts. Expression was enhanced with the addition of irradiated Flavobacterium columnare (7.0 x 106 cells/ml) or Escherichia coli LPS (125 μg/ml). Quantitative RT-PCR showed GFP mRNA reached maximum levels 24 h after bacterial challenge in fibroblast cells, and at 10-12 h after LPS challenge in fibroblasts and leukocytes. The number of fibroblasts expressing GFP increased by 0.8%, and the average of green fluorescence intensity increased by 52.8%, whereas the increase in leukocytes was 0.13% in cell number and 3.4% in fluorescence intensity. These results suggest that the transcription of the Cec B promoter in channel catfish cells exhibited an inducible pattern and could be placed under the control of the immune system (in vivo). The mechanisms for endogenous activation of the Cec B promoter and for production of gfp RNA in unchallenged cells remain to be studied.

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