| 研究概要 |
We performed the transfection of CASPAR transcripts CASPAR5 and CASPAR6 into THP1 cells, which we expect are targeting the STAT6 transcription factor. Indeed we saw a decrease of 20-30% in the expression of STAT6 upon transfection of CASPAR5. However we also saw a decrease in the expression of STAT6 after the transfection of GFP (green fluorescent protein). We then tried a different transfection protocol using lipofectamine, Our hypothesis is that the changes in gene expression after GFP transfection are due to an interferon response, and that the transfection of foreign DNA into THP1 cells is causing them to differentiate from a monoblast state to a macrophage state. This was confirmed by measuring the expression level of the MYB and GFI1 genes, which were similarly affected by the transfection. We then tried to use a different transfection protocol based on a lentivirus vector. Here, we did not find a strong interferon response in THP1 cells upon lentivirus transduction, and we are now preparing the lentivirus constructs containing the CASPAR transcripts. In parallel we are also preparing to knock down the endogenously expressed CASPAR transcripts using antisense oligos as a complementary approach to understanding the function of CASPAR transcripts in cellular regulation.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
4: 遅れている
理由
During the execution of this project, the laboratory member who was performing the wet-lab experiments described in the project proposal was offered a position at a different laboratory. This person decided to leave RIKEN, which caused some delay in this project. Most of the remaining budget will be used for transcriptome sequencing. This is the final step in the project, and by far the most expensive, which is why a rather large amount of budget is left for the final year.
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| 今後の研究の推進方策 |
We will finish producing the lentivirus constructs containing the CASPAR transcripts, transfect them into THP1 cells, and measure the expression level of the sense gene by qPCR to assess their regulatory effect. We will also attempt to knock down CASPAR transcripts using the antisense oligos, and again measure the expression level of the sense gene by qPCR. If we see an effect in either case, we will proceed with genome-wide transcriptome profiling using CAGE. If not, we will use the remaining budget for sequencing of capped short RNAs of the typical size of CASPAR transcripts during a time course of THP1 differentiation to at least produce a global map of CASPAR transcripts and their dynamics.
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| 次年度の研究費の使用計画 |
After starting this project, the laboratory member who was performing the wet-lab experiments was offered a position at a different laboratory and left our laboratory. This caused some delay in this project. A postdoctoral researcher at our laboratory is now performing these wet-lab experiments. However, as he cannot commit full-time to this project, progress has been slower than planned. Also, CASPAR transfection into THP1 cells using plasmids resulted in a strong interferon response, so we decided to try lentivirus transfection instead.
We are now preparing the entry clones needed to create the CASPAR-containing lentivirus for transfection into THP1 cells, which will be followed by transcriptome sequencing. Of the remaining budget, 127,633 yen will be used to cover lentivirus production, cell culture, and qPCR validation of CASPAR expression, and 890,000 yen for deep sequencing of the transcriptome after CASPAR transfection (416,000 yen for CAGE library preparation and 474,000 yen for sequencing using the Illumina HiSeq2500 sequencer).
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