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

这项提案的长期目标是定义分子事件 控制正常和白血病的早幼粒细胞特异性“程序”， 细胞，并定义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， CG基因仅在早幼粒细胞中表达; 或在人CG基因附近含有早幼粒细胞靶向信号。 我们 我打算利用这些信息来进一步表征 早幼粒细胞，通过以下具体目的： 1. 我们将定义靶向表达的调控DNA序列， CG对转基因小鼠中的早幼粒细胞的影响。 我们已经证明，一个6.0 kb的 含有人CG基因的片段特异性靶向骨髓 转基因小鼠中的前体。 我们将剖析其中的序列 片段以定义早幼粒细胞靶向元件，并鉴定 与这些元素特异性相互作用的蛋白质。 2. 我们将研究急性早幼粒细胞白血病（APL）的生物学， 将PML-RAR α和RAR α-PML cDNA靶向小鼠早幼粒细胞， CG衍生的靶向序列。 我们创造了一种新的目标载体 含有来自6.0 kb CG转基因的所有序列。 我们将使用 E2 A-PBX-1 cDNA以确认靶向载体的准确性， 确定是否可以在体内转化早幼粒细胞。 然后我们将 靶向由与APL相关的t（15;17）易位产生的cDNA。 一 PML-RAR α cDNA和相互的RAR α-PML cDNA将被单独地表达。 靶向转基因小鼠的早幼粒细胞，以确定 这些cDNA能够引起早幼粒细胞分化的阻滞 或明显的APL。 表达转基因品系将彼此繁殖， 确定两个转基因是否协同产生APL。 的 转基因动物将被仔细地描述，以创建模型， 研究这种疾病。 3. 我们将建立小鼠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， 胚胎干细胞和笼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 上述定义的“APL”小鼠发生DIC，我们将它们繁殖到CG-/-小鼠中。 背景，以进一步明确这一作用。

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.