HOMEOBOX GENES IN HEMATOPOIETIC DIFFERENTIATION AND MATURATION

造血分化和成熟中的同源盒基因

• 批准号: 6110523
• 负责人: JUDITH Cheryl GASSON
• 金额: $ 20.04万
• 依托单位: CHILDREN'S HOSPITAL OF LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6110523
• 关键词: Drosophilidae; Xenopus; antisense nucleic acid; colony stimulating factor; embryogenesis; flow cytometry; gene expression; gene targeting; genetic transcription; hematopoiesis; hematopoietic growth factor; homeobox genes; polymerase chain reaction; representational difference analysis; tissue /cell culture; transfection /expression vector

(Adapted from the applicant's abstract) The goal of these studies is to determine how the multilineage hematopoietic growth factor, granulocyte- macrophage colony-stimulating factor (GM-CSF), stimulates the proliferation and maturation of progenitor cells into specific myeloid elements. Our studies will focus on a class of genes known to play an important role in coordinating embryogenesis and differentiation in Drosophila and Xenopus, the homeobox genes. These genes encode transcription factors that contain a highly conserved 60-amino acid DNA- binding motif known as the homeodomain. Homeobox genes can both positively and negatively regulate the transcription of other genes, and consequently, they play critical roles in anteriorposterior axis formation, segmentation, and orchestrate the coordinated expression of genes required to generate complex structures. The focus of this grant application is to identify the homeobox genes that are involved in myeloid differentiation and to elucidate how interaction of GM-CSF with the myeloid progenitor cells affects the expression and action of these homeobox genes. Specific Aim 1 - Identify and characterize homeobox genes involved in human myelopoiesis. Reverse transcription polymerase chain reaction (RT-PCR), using degenerate primers recognizing the homeodomain, is employed to identify the homeobox genes expressed in normal bone marrow progenitors stimulated by GM- CSF, in semi-solid media, to undergo two or three cell divisions. The expression of specific homeobox genes in myeloid progenitors will be confirmed using quantitative RT-PCR (and specific primers) of fluorescence-activated cell sorted (FACS) bone marrow subsets. Evidence supporting a functional role for these homeobox genes will be obtained using antisense oligonucleotides (ODN) to block their expression in in vitro assays. We have successfully employed these strategies to identify three HOX genes, and thus far, have obtained biological data supporting the role of two of them in myelopoiesis. Specific Aim 2 - Employ biological assays to determine the functional role of specific homeobox genes in human myelopoiesis. Retroviral vectors will be used to enforce expression of specific homeobox genes in hematopoietic cell lines and primary hematopoietic progenitor cells. Cell lines will be assayed for their responses to multiple differentiation inducers. Transduced CD34+ progenitor cells will be assayed in long-term culture assays for their differentiation and proliferative potentials. Specific Aim 3 - Identify target genes that are regulated by specific homeobox proteins during myelopoiesis. Homeobox genes identified in Aim 1 and shown to play a physiologic role in myelopoiesis in studies described in Aim 2 will be used to isolate target genes. Inducible plasmid vectors directing the expression of a specific HOX gene will be stably transfected into a model hematopoietic cell line; "representational difference analysis" (RDA) will be used to identify target genes that are either activated or repressed by the expression of this homeobox gene.

（根据申请人的摘要改编）这些研究的目的是 确定多素造血生长因子如何，粒细胞 - 巨噬细胞刺激因子（GM-CSF）刺激 祖细胞扩散和成熟到特定的髓样 元素。我们的研究将集中于一类已知的基因 在协调胚胎发生和分化中的重要作用 果蝇和爪蟾，同源物基因。这些基因编码 包含高度保守的60氨基酸DNA-的转录因子 绑定的主题称为同源域。同源基因都可以 积极和负调节其他基因的转录，并 因此，它们在前轴上起关键作用 形成，分割和编排协调的表达 产生复杂结构所需的基因。这笔赠款的重点 应用是确定涉及的同源基因 髓样分化并阐明GM-CSF的相互作用如何 髓样祖细胞影响这些细胞的表达和作用 同源基因。 特定目的1-识别和表征涉及的同源基因基因 人脊髓卵。逆转录聚合酶链反应 （RT-PCR）使用识别同源域的退化引物为 用于识别在正常骨髓中表达的同源基因 GM-CSF在半固体介质中刺激的祖细胞，以进行两种 或三个细胞分裂。特定同源基因在 将使用定量RT-PCR确认髓样祖细胞（和 荧光激活的细胞（FACS）骨的特定引物） 骨髓子集。支持这些功能作用的证据 同型基因将使用反义寡核苷酸（ODN）获得 阻止其在体外测定中的表达。我们已经成功 采用这些策略来识别三个HOX基因，到目前为止 获得了支持其中两个在 骨髓。特定目的2-使用生物学测定来确定 特定同源基因在人脊髓卵中的功能作用。 逆转录病毒载体将用于强制执行特定的表达 造血细胞系和主要造血的同源基因基因 祖细胞。细胞系将通过其对 多个分化诱导剂。转导CD34+祖细胞 将在长期文化分析中分析其差异化 和增殖潜力。特定目标3-识别目标基因 在骨髓卵中，特定的同型蛋白质调节。 在AIM 1中鉴定出的同源基因并显示出生理角色 在AIM 2中描述的研究中，在骨髓卵中将用于分离 靶基因。引导A表达的诱导质粒向量 特定的HOX基因将被稳定转染到模型造血基因 细胞系； “代表性差异分析”（RDA）将用于 识别被激活或抑制的靶基因 该同源基因的表达。