Inducible expression of green fluorescent protein within channel catfish cells by a cecropin gene promoter.
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 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 ¿10(6) cells/ml) or Escherichia coli LPS (125microgram/ml). Quantitative RT-PCR showed GFP mRNA reached maximum levels 24h after bacterial challenge in fibroblast cells, and at 10-12h 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.
Department of Veterinary Science, Louisiana Agricultural Experiment Station, Louisiana State, University Agricultural Center, Baton Rouge, LA 70803, USA.
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Zhang
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Tiersch
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Cooper
D000818:Animals; D002460:Cell Line; D004274:DNA, Recombinant / Q000235:genetics; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000235:genetics; D049452:Green Fluorescent Proteins; D007059:Ictaluridae / Q000235:genetics*; D007202:Indicators and Reagents / Q000378:metabolism; D019476:Insect Proteins / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D012313:RNA / Q000032:analysis / Q000235:genetics
D004274:DNA, Recombinant; D007202:Indicators and Reagents; D019476:Insect Proteins; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D012313:RNA; C031162:cecropin B protein, Insecta