REGULATION OF HUMAN CATHEPSIN G GENE IN NORMAL AND LEUKEMIC BLOOD CELLS

这项提议的长期目标是定义分子事件 它控制着正常人和白血病患者的早幼粒细胞特异性“程序” 细胞，并确定早幼粒细胞特异性酶的功能 组织蛋白酶G(CG)。我们的实验室已经确定，小鼠和人类 CG基因只在早幼粒细胞中表达； 或在人类CG基因附近含有早幼粒细胞靶向信号。我们 意愿利用这一信息来进一步描述 早幼粒细胞，通过以下特定目的： 1.我们将定义针对其表达的调控DNA序列 在转基因小鼠中将CG转化为早幼粒细胞。我们已经证明了6.0kb 含有人CG基因的片段是针对髓系细胞的 转基因小鼠体内的前体细胞。我们将仔细分析其中的序列 片段以定义早幼粒细胞靶向元件，并鉴定 与这些元素特别相互作用的蛋白质。 2.对急性早幼粒细胞白血病(APL)的生物学研究 靶向PML-RARpha和RARpha-PML cDNA的小鼠早幼粒细胞 CG衍生的靶向序列。我们创造了一种新的靶向载体 包含来自6.0kb CG转基因的所有序列。我们将使用 确定靶向载体准确性的E2A-PBX-1 cDNA和TO 确定早幼粒细胞是否可以在体内转化。到时候我们会的 与APL相关的t(15；17)易位所产生的靶cDNA。一个 PML-RARpha cDNA和一个相互的RARpha-PML cDNA将分别 以转基因小鼠中的早幼粒细胞为靶点，以确定 这些cDNA能够阻止早幼粒细胞分化。 或公开的APL。表达转基因的品系将相互培育以 确定这两种转基因是否协同作用产生APL。这个 转基因动物将被仔细描述，以创建用于 对这种疾病的研究。 3.建立小鼠CG功能丧失模型。我们将使用 在小鼠CG基因中产生靶向突变的标准策略 胚胎干细胞，以及cg-/-小鼠。将对这些小鼠进行检查 体外和体内中性粒细胞和巨噬细胞功能缺陷。CG 可能在导致与人类APL相关的DIC综合征中发挥作用；如果 上面定义的“APL”小鼠发生DIC，我们将把它们培育成CG-/- 进一步定义这一角色的背景。

The long-term goals of this proposal are to define the molecular events that control the promyelocyte-specific "program" of normal and leukemic cells, and to define the functions of the promyelocyte-specific enzyme cathepsin G (CG). Our laboratory has determined that the murine and human CG genes are expressed only in promyelocytes; cis-acting sequences within or near the human CG gene contain the promyelocyte-targeting signals. We intend to use this information to further characterize the biology of promyelocytes, via the following Specific Aims: 1. We will define the regulatory DNA sequences that target expression of CG to promyelocytes in transgenic mice. We have shown that a 6.0 kb fragment containing the human CG gene is targeted specifically to myeloid precursors in transgenic mice. We will dissect the sequences within this fragment to define the promyelocyte targeting elements, and identify proteins that interact specifically with these elements. 2. We will study the biology of acute promyelocytic leukemia (APL) by targeting PML-RARalpha and RARalpha-PML cDNAs to murine promyelocytes with CG-derived targeting sequences. We have created a novel targeting vector containing all of the sequences from the 6.0 kb CG transgene. We will use an E2A-PBX-1 cDNA to confirm the accuracy of the targeting vector, an to determine whether promyelocytes can be transformed in vivo. We will then target cDNAs created by the t(15;17) translocation associated with APL. A PML-RARalpha cDNA, and a reciprocal RARalpha-PML cDNA will be individually targeted to promyelocytes in transgenic mice to determine whether either of these cDNAs is capable of causing a block in promyelocyte differentiation or overt APL. Expressing transgenic lines will be bred to one another to determine whether the two transgenes synergize to produce APL. The transgenic animals will be carefully characterized to create models for the study of this disease. 3. We will create a loss of function model for murine CG. We will use standard strategies to create a targeted mutation in the murine CG gene in embryonic stem cells, and crate CG-/-mice. These mice will be examined for defects in neutrophil and macrophage functions in vitro and in vivo. CG may play a role in causing the DIC syndrome associated with human APL; if the "APL" mice defined above develop DIC, we will breed them into the CG-/- background to further define this role.