| 研究実績の概要 |
Our project focuses on understanding the biological role of CASPARs, a novel family of capped short transcripts that overlap coding genes near their promoter region in the antisense orientation. As overexpression of CASPARs by lentivirus transfection of CASPARs in the THP-1 cells was unsuccessful because of the immune response of the cells, in this fiscal year we tried instead to knockdown the CASPAR transcripts using antisense oligos (ASOs), using 2 ASOs for each of the 13 CASPARs. For most knockdowns, we found an upregulation or downregulation of the sense gene (as measured by qPCR), suggesting that the CASPARs are functional. We developed a strand-specific qPCR protocol to verify the knockdown efficiency of the CASPAR itself. The strand-specific qPCR confirmed the knockdown efficiency for three CASPARs. We will now proceed to perform CAGE expression profiling on the knockdown samples to assess the genome-wide transcriptome response of the CASPAR knockdown.
We also subjected THP-1 cells to PMA stimulation, causing the THP-1 monoblast cells to differentiate into monocytes, and extracted RNA at several time points during this differentiation process. We produced CAGE libraries from these RNA samples, using a novel protocol under development in our laboratory to enrich the CAGE library for short transcripts. We performed paired-end sequencing of a test library on a MiSeq sequencer, confirming that this protocol can detect CASPAR transcripts. Next, we will apply single-end sequencing to these libraries to measured the CAGE expression dynamics of the CASPARs.
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