2018 Fiscal Year Annual Research Report
Role of inflammation in initiation and progression of clonal hematopoiesis
|Research Institution||Kumamoto University |
滝澤 仁 熊本大学, 国際先端医学研究機構, 特別招聘教授 (10630866)
|Foreign Research Fellow
FAKRUDDIN MD. 熊本大学, 国際先端医学研究機構, 外国人特別研究員
|Project Period (FY)
2018-10-12 – 2021-03-31
|Keywords||clonal hematopoiesis / inflammation|
|Outline of Annual Research Achievements
Lifelong blood production is maintained by self-renewing and multi-lineage differentiating hematopoietic stem cell (HSC). Inflammation associated with infection and aging increases the division of hematopoietic stem cells and decreases its cellular function. The advanced sequencing technology has demonstrated that gradual accumulation of genetic mutations in HSCs alters clonal hematopoiesis triggering hematological disorders and leukemia. Here, we will study how inflammation is involved in the process using clonal hematopoietic tracer mice that we will newly generate.
|Current Status of Research Progress
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Objective 1) Generation of animal model for clonal barcoded hematopoiesis
(A) Establishment of efficient genome barcode amplification method
Method based on Linker Mediated PCR for efficient genome barcode amplification has been established and optimized using cell line (BA/F3 and FDC-P1) transfected with Sleeping beauty transposon element. The method can detect both clone number and clone size simultaneously which provide it advantage over other reported method. The method can distinguish expanded clones with low number of cells (as low as 104) and low amount of target genomic DNA (as low as 500 ng). Efficiency of transposon integration did not have any effect on the sensitivity of the method. The method is ready to employ in in vivo system.
(B) Generation of clonal hematopoietic model mice
Mice with multiple copy of transposon cassette (IR-CAG-GFP-IR) under CAG promoter, termed as Poly-IR mice, has been generated already. Mice that conditionally express Hyper Sleeping Beauty (HSB), a transposase under ROSA26 locus has been cloned at ES level successfully. The mice will be generated, and will be crossed with both HlfCreERT2 mice to express HSB conditionally, and the mice that express multiple copies of GFP with HSB recognition. Upon crossing tamoxifen infection that activate HSB, HSB will be activated by administration of tamoxifen, and mice expressing GFP by inserting CAG-GFP-IR into other chromosomal loci will be prepared.
|Strategy for Future Research Activity
Objective 2) Clonal hematopoiesis under steady- or inflammatory stress
Mononuclear cell fraction will be sorted from the peripheral blood of mouse adults after HSB induction, using the mouse of Objective 1-B), and target 1-A) and the number and size of clones will be determined by the method developed in this study. The hematopoietic stem cell / progenitor cell fraction of the bone marrow will be re-evaluated after a certain period of time.
Once these technologies and experimental materials are established, it will provide useful tools not only in the field of hematopoietic stem cells but also in other biomedical research fields such as stem cell and cancer biology. The knowledge obtained from this study is expected to provide contribution towards establishment of preventive methods for pre-leukemia initiation and progression.
Research Products (1 results)