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.
Zhang
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Department of Veterinary Science, Louisiana Agricultural Experiment Station, Louisiana State, University Agricultural Center, Baton Rouge, LA 70803, USA.
Tiersch
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Cooper
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RK
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