Role of inflammation in initiation and progression of clonal hematopoiesis

世界各地的许多实验室一直致力于开发利用不同机制进行谱系追踪的新方法。但是，这些方法无法提供失调克隆和扩增克隆的克隆大小信息，因此我们设计了一种同时确定克隆性和克隆大小的方法。我设计、开发和评估了一种新的克隆追踪方法，该方法在转染了睡美人转座子载体的细胞系中进行了克隆追踪，数据分析表明，该方法可以检测到大约500个克隆（包括扩增和非扩增），并具有各自的克隆大小。整合位点的分布分析显示转座子在所有染色体上的整合是随机分布的。我还用细胞数评估了该方法，低至10000个细胞，该方法工作良好，可以检测到足够的克隆及其各自的克隆大小。为了在体内应用这种方法，需要新的小鼠品系。因此，我还培育了两种新的小鼠品系——含有转座酶的Rosa26-Hyper sleeping beauty （HSB）和染色体沉默位点含有多拷贝IR转座子的polyIR小鼠。如果在小鼠模型中成功评估该方法，它将不仅在血液学领域，而且在其他生物医学研究领域提供有用的工具来追踪谱系到单细胞分辨率。从这项研究中获得的知识有望为建立白血病前期开始和进展的预防方法提供贡献。

Many labs around the world has been working to develop novel methods for lineage tracing employing different mechanisms. But, these methods cannot provide the information on clone size of the dys-regulated and expanded clones, which led us to design this study to develop a method to determine both clonality and clone size simultaneously.I designed, developed and evaluated the novel clonal tracing method in cell line transfected with sleeping beauty transposon vectors and analysis of data revealed that the method can detect around 500 clones (both expanded and non-expanded) with their respective clone size. Distribution of integration site analysis showed random and stochastic distribution of transposon integration in all the chromosomes. I also evaluated the method with cell number and with as low as 10000 cells, the method work well and can detect enough clones with their respective clone size. To apply this method in vivo, novel strains of mice were needed. So, I also generated two novel strains of mice- Rosa26-Hyper sleeping beauty (HSB), which contains transposase enzyme and polyIR mice, which contains multiple copy of IR transposon in silent locus of chromosome.Upon successful evaluation of the method developed in this study in mouse model, it will provide useful tools not only in the field of hematology but also in other biomedical research fields to trace lineage to single cell resolution. The knowledge obtained from this study is expected to provide contribution towards establishment of preventive methods for pre-leukemia initiation and progression.