| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2005: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2004: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Research Abstract |
The Zebrafish, Danio rerio, is a small teleost that has become a laboratory favorite because its embryos are transparent, allowing geneticists to in effect see genes at work in the developing fish. However, lack of an appropriate cell culture system in which the genetic modification of the cell can be transmitted into the germ-line has prevented the development of reverse genetics approaches. We have recently developed techniques to make genetically modified zebrafish using sperm cells grown from premeiotic germ cells in vitro. Here we tried to develop the RNA interference (RNAi) protocol for studying gene functions in zebrafish by using in vitro-cultured sperm.
Because the efficiency of RNA interference depends on the sequence of small interference RNA (siRNA), we examined the effect on the mRNA amount and the phenotype by injecting siRNA of shh or gfp into 1-cell stage embryos. One shh siRNA among 4 sequences and the gfp siRNA that has already reported decreased the corresponding mRNA
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. Although the gfp siRNA did not suppress the cellular fluorescent in the transgenic fish, the shh siRNA showed the mutant phenotype with slight off-targeting effect. The pseudotyped retroviral vectors of the DNA-based short hairpin RNA (shRNA) are currently constructing. On the other hand, the analysis of siRNA sequence by the injection into embryos required labor works and a lot of times. Thus, we decided establishment of cultured cells to determine specific suppression of a target gene by transfection of several possible designed siRNAs. It is easy to grow cultured cells and analyze the gene product, which saves time. A cell line derived from the midblastula stage still expressed several appropriate genes. The condition of electroporation for the cells was also determined and performed the 30-40% efficiency of transfection. By establishment of various cell lines from different developmental stages and tissues, it will be possible to determine the best sequence of siRNA in vitro easily. After construction of the viral vector of shRNA, we will use it to infect spermatogonia, and then make a transgenic zebrafish from in vitro-cultured sperm. Less
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