| 研究実績の概要 |
The chromatin remodeler ATRX is frequently mutated in neuroblastoma (NB) and in other pediatric tumors. Recent study confirmed that some NB tumors appear to maintain telomere length by activating the ALT pathway which is a telomerase independent mechanism of telomere length maintenance, and ALT activity was associated with significantly reduced overall survival. A unifying feature of these tumors is mutations in ATRX. Unfortunately, the molecular mechanism of ALT activity is unknown. Therefore, our primary objectives is to analyzing the link between ATRX loss of function and ALT pathway in NB, which will be remarkable clue for the development of ALT-related targets, and may contribute to the individualized treatment for high risk NB cases.
To date, we used the CRISPR/Cas9 system, which is the robust genome editing tool for genetic loss-of-function (LoF) studies of gene-of-interest in a wide variety of organisms. First, We have developed the ATRX knockout (KO) clones in MYCN amplified (NGP) and MYCN single copy (NB-69) NB cells using CRISPR-Cas9 technology. The clones proliferate slowly compared to control and become differentiated.
Interestingly, our qPCR assay showed that KO clone has the lower telomere content than ALT positive SK-N-FI cells. Furthermore, microarray gene expression analysis also revealed that KO clones have the different gene expression signature than ALT+ ve cell lines. In conclusion, our data indicate that loss of ATRX alone is not enough to activate ALT in NB cells. Further studies are aimed at understanding how ATRX affects the ALT mechanism in NB.
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| 次年度使用額が生じた理由 |
We have done some minor change in our research plan. Initially we have decided to use CRISPR/ Cas9 system in some more cells. Because of cells unavailability, we couldn't do. And also some experiment materials (example) we couldn't buy last fiscal time because of unavailability.
This year, we are going to use more advanced CRISPR/ Cas9 system for more efficiently knock out the targeted gene. And we will add the remaining amount in new fiscal year to analyze the biological properties of ATRX knock out cells.
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