| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2004: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2003: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Research Abstract |
The transfer of genes of interest is a useful method for studying placental biology. Recombinant adenovirus (Ad) vector is run efficient vector for Transgene ne expression. An interaction between the fiber of Ad and the coxackievirus and adenovirus receptor (CAR) on the cell membrane is the first step in infection. We developed fiber-modified Ad vectors and showed that they improved transgene activity un several cell Inns when compared to wild-type vector: Subsequently, we investigated the ability of three fiber-modified Ad vectors to transduce human choriocarcinorna cell lines; (JEG-3, JAR and BeWo) used as in vitro models of human placenta, and rat trophoblast cell lines (Rcho-1, TR-TBT 18d-1 and TR-TBT 18d-2) We compared the transgene efficiency of wild-type Ad-L2 vector, Ad-RGD(HI)-L2 vector containing an Arg-Gly- Asp motif, Ad-K7(C)L2 vector containing a 7 tandem lysine motif, and Ad-RGD(HI)K7(C)-L2 vector containing both motifs in the fiber. The luciferase gene as a reporter gene
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was used. In the human and rodent trophoblast cell lines, Ad-RGD(HI)-L2 had the greatest infectious potential, followed by Ad-RGD(HI)K7(C)-L2. Ad-K7(C)L2 fund Ad-L2. Compared to the amount of luciferase produced by wild-type vector Ad-RGD(HI)-L2 mediated 8-fold the amount of luciferase in JEG-3 cells. 14-fold in JAR cells, 77-fold im BeWo cells, 5-fold in Rcho-1,19-fold in Tr-TBTa8d-1 and 15-fold in TR-TBT 18d-2. These results indicate that Ad-RGD(HI) is a potential recombinant Ad vector for transgene expression in some trophoblast cell lines. Conventional Ad type 5 vectors have a narrow range of tropism and are limited by the size of the transgene that can be packaged. To overcome these limitations, we developed an Ad vector (Ad5/35 vector) containing a chimeric Ad type 5 amd 35 fiber protein. We evaluate the ability of the Ad5/35 vector to transfer genes into human trophoblast cell lines (JAR, JEG-3 and BeWo cells) above mentioned. It was found thatt expression of CD-46, which are recptors for Ad5/35 vector, are higher than that of CAR in all 3 trophoblast cell lines, as determined by flow cytometry. Sunsequently, we compared the trnasducing activity of Ad5 vector and Ad5/35 vector that each epressed luciferase as a reporter gene. Ad5/35 vector had superior gene transfer activity than the conventional Ad vector in all three trophoblast cell lines (1.8-fold in JAR cells, 5-fold in BeWo cells, 6-fold in JEG-3 cells). This, Advector that contains chinnerictype 5 and 35 fiber protein can be a powerful tool for gene trnasfer experiments in human trophoblast cell lines. Less
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