| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2000: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1999: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
The avidin-biotin binding is very specific and strong. The objects of this project are to fix biotinylated drugs on the endothelia by biotinylating directly the cells and giving them avidin molecules. In vivo study using rabbit kidneys, we injected a biotinylating reagent with NHS-ester, which react with cellular proteins, from a vascular catheter inserted into a renal artery. After five-minute incubation, we applied avidin solution and, subsequently, fluorescence-labeled biotin solution. A histological study showed that more than 80% of glomeruli were stained with fluorescence, which meant that biotinylated materials can be fixed on the endothelia in vivo by the present procedure. The same result was obtained in vitro using cultured endothelial cells. However, when we used biotinylated drug solutions whose concentrations were lower than 10^<-8>M, they were not detected in the cells. So we first mixed an avidin solution and a biotinylated drug (r-phycoerythrin-biotin (r-PE-biotin) solution in the ratio 100:1 (in mole) to make avidin-biotinylated drug complex. We applied the complex to the biotinylated endothelia, and found that r-PE-biotin was fixed on the cells. Based on the result, we made biotinylated double strand (ds) DNA, which had the gene of green fluorescence protein (GFP), and mixed it (10^<-9> and 10^<-8>M) with excessive volume of avidin solution. We applied the biotinylated ds-DNA- avidin complex to the biotinylated cells in logarithmic growth. After two-day incubation, we found the cells expressing GFP. We believe the present procedure is a novel method for gene transfer.
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