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采用不同机制的谱系追踪新方法。但是，这些方法不能提供关于失调和扩增的克隆的克隆大小的信息，这导致我们设计本研究以开发同时确定克隆性和克隆大小的方法。开发并评估了在用睡美人转座子载体转染的细胞系中的新型克隆追踪方法，数据分析显示该方法可以检测到约500个克隆（扩增的和未扩增的）具有它们各自的克隆大小。整合位点的分布分析表明转座子在所有染色体上的整合是随机和随机分布的。我还用细胞数和低至10000个细胞的方法进行了评价，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，我还产生了两种新的小鼠品系-Rosa 26-Hyper sleeping beauty（HSB），其含有转座酶和polyIR小鼠，其在染色体的沉默位点中含有多拷贝的IR转座子。它将不仅在血液学领域而且在其它生物医学研究领域提供有用的工具以追踪谱系到单细胞分辨率。从本研究中获得的知识有望为建立预防白血病前期启动和进展的方法做出贡献。

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.