Effect of MKL1 on murine embryonic megakaryocytopoiesis

描述（由申请方提供）：急性巨核细胞白血病的特征是巨核细胞分化受阻和增殖不当。已发现与AMKL婴儿相关的1;22染色体易位涉及白血病发生中的融合基因产物。融合基因由在读码框中融合并位于RBM 15下游的全长MKL 1组成。本实验室主要研究MKL 1（一种心肌蛋白相关转录因子）在巨核细胞生成调控中的正常和异常功能。我们建议在小鼠胚胎干细胞（ES）中研究MKL 1，试图模拟发育性造血，并进一步了解新生儿系统中的疾病。我们将测试MKL 1的强制表达和错误表达是否对巨核细胞分化有影响。为了检验MKL 1的强制表达促进小鼠ES细胞分化的假设，我们将开发诱导型MKL 1 ES细胞系并分析其MK分化。此外，我们将进行研究，以确定巨核细胞分化中对MKL 1和/或MKL 2的需求。为此，我们将构建表达显性失活MKL 1或针对MKL 1和/或MKL 2的shRNA的诱导型ES细胞克隆。为了确定对巨核细胞生成的影响，我们将使用CFU-Mk（功能测定）、流式细胞术分析（表型测定）和细胞离心涂片（形态学测定）。在细胞培养研究的同时，我们将对MKL 1-/-小鼠进行更多的造血集中分析，并进一步操作动物模型以辨别MKL 1在巨核细胞生成中的作用。这项工作的目标是建立一个可以翻译成人类细胞的小鼠系统，用于研究由1;22易位引起的AMKL。这一转变将为基于治疗的研究创造机会，并最终为患病个体提供更明智和更精确的治疗。

DESCRIPTION (provided by applicant): Acute Megakaryoblastic Leukemia is characterized by the blocked differentiation and improper proliferation of megakaryocytes. The 1;22 chromosomal translocation that has been found to associate with AMKL infants implicates the fusion gene product in leukemiogenesis. The fusion gene consists of full length MKL1 fused in frame and downstream of RBM15. Our lab focuses on the normal and aberrant functioning of MKL1, a myocardin related transcription factor, in the regulation of megakaryocytopoiesis. We propose to study MKL1 in murine embryonic stem (ES) cells in an attempt to mimic developmental hematopoiesis and further understand the disease in the neonatal system. We will test if enforced and misexpression of MKL1 has an effect on megakaryocyte differentiation. To test the hypothesis that enforced expression of MKL1 promotes mouse ES cell differentiation we will develop an inducible MKL1 ES cell line and analyze it for MK differentiation. In addition, we will conduct studies to determine the requirement for MKL1 and/or MKL2 in megakaryocyte differentiation. To do this, we will construct inducible ES cell clones expressing a dominant negative MKL1 or shRNA against MKL1 and/or MKL2. To determine the effects on megakaryocytopoiesis we will use CFU-Mk (a functional assay), flow cytometric analysis (a phenotypic assay), and cytospin preparations (a morphological assay). Concurrent with the cell culture investigations, we will conduct a more hematopoietic focused analysis of MKL1-/- mice and further manipulate the animal model to discern the role of MKL1 in megakaryocytopoiesis. The goal of the work is to create a mouse system that can be translated into human cells for the study of AMKL caused by the 1;22 translocation. This transition will create opportunities for therapy based research and ultimately more informed and precise treatments for afflicted individuals.