Effect of MKL1 on murine embryonic megakaryocytopoiesis

描述（申请人提供）：急性巨核母细胞白血病以巨核细胞分化受阻、增殖异常为特征。1;22染色体易位已被发现与AMKL婴儿相关，暗示融合基因产物在白血病发生中。融合基因由全长MKL1和RBM15的下游组成。本实验室主要研究心肌素相关转录因子MKL1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MKL1，试图模拟发育造血，并进一步了解新生儿系统中的疾病。我们将测试MKL1的强制和错误表达是否对巨核细胞分化有影响。为了验证强制表达MKL1促进小鼠胚胎干细胞分化的假设，我们将建立一个诱导MKL1胚胎干细胞系，并对其进行MK分化分析。此外，我们将开展研究以确定巨核细胞分化对MKL1和/或MKL2的需求。为此，我们将构建可诱导的ES细胞克隆，表达MKL1和/或MKL2的显性阴性MKL1或shRNA。为了确定对巨核细胞生成的影响，我们将使用CFU-Mk（一种功能分析）、流式细胞分析（一种表型分析）和细胞自旋制剂（一种形态分析）。在进行细胞培养研究的同时，我们将对MKL1-/-小鼠进行更侧重于造血的分析，并进一步操作动物模型以确定MKL1在巨核细胞生成中的作用。这项工作的目标是创建一个可以转化为人类细胞的小鼠系统，用于研究由1；22易位。这一转变将为基于治疗的研究创造机会，并最终为患者提供更明智、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.