Inducible Expansion of Hematopoietic Stem Cells

造血干细胞的诱导扩增

基本信息

批准号：
7140250
负责人：
Donald F. Doyle
金额：
$ 20.29万
依托单位：
GEORGIA INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-07-01 至 2008-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7140250
关键词：
Retroviridae binding sites biotechnology gene expression genetic transduction genetically modified animals hematopoietic stem cells hematopoietic tissue transplantation laboratory mouse protein structure function recombinant DNA recombinant proteins stem cell transplantation technology /technique development transfection transfection /expression vector

项目摘要

DESCRIPTION (provided by applicant): Within the community of scientists who study the biology and application of stem cells there is a need for the controlled expression of specific gene sequences. Our overall objective is to develop recombinant gene transfer systems with two features: tight control of transgene expression and inducible transgene expression. Specifically, we will develop vectors that expand populations of multipotent hematopoietic stem cells (HSCs) both ex vivo and in vivo while maintaining their undifferentiated state. Investigators have tested a variety of strategies to accomplish this aim, but success has been hampered by the complexity of reagents required to control transgene expression. We have generated a fusion protein that combines a genetically engineered ligand binding domain specific for a synthetic "near drug" and a DMA binding domain specific for a DNA sequence not found in the mammalian genome (i.e. the Gal4 response element). By cloning cDNA sequences downstream of the response element we have achieved remarkable control over transcription of several cDNA sequences. Our strategy has many advantages over currently available systems, including exquisitely tight control over transgene expression and high selectivity of our engineered ligand binding domain. Using this system, we have designed first generation retroviral constructs that will allow controlled transgene expression in genetically engineered cells. The goals of this proposal are to determine the extent that control of proviral sequences can be genetically engineered into retroviral constructs and to test the effectiveness of our inducible systems to regulate the controlled expression of cDNA sequences that regulate the growth of multipotent HSCs. Our first aim is to develop and assess the effectiveness of orthogonal promoters that express reporter genes (luciferase and GFP) as well as HoxB4, which has clearly been shown to be important in the expansion of HSCs. Our second aim is to test regulated expression of HoxB4 in HSCs transduced with our inducible vectors to enforce HSC expansion in vivo.

描述（由申请人提供）：在研究干细胞生物学和应用的科学家群体中，需要特定基因序列的受控表达。我们的总体目标是开发具有两个特征的重组基因转移系统：转基因表达的严格控制和可诱导的转基因表达。具体来说，我们将开发能够在离体和体内扩大多能造血干细胞（HSC）群体的载体，同时保持其未分化状态。研究人员测试了多种策略来实现这一目标，但控制转基因表达所需试剂的复杂性阻碍了成功。我们已经生成了一种融合蛋白，它结合了对合成“邻近药物”特异的基因工程配体结合结构域和对哺乳动物基因组中未发现的 DNA 序列特异的 DMA 结合结构域（即 Gal4 反应元件）。通过克隆反应元件下游的 cDNA 序列，我们已经实现了对几个 cDNA 序列转录的显着控制。与目前可用的系统相比，我们的策略具有许多优势，包括对转基因表达的严格控制以及我们工程配体结合域的高选择性。使用该系统，我们设计了第一代逆转录病毒构建体，该构建体将允许在基因工程细胞中控制转基因表达。该提案的目标是确定对原病毒序列的控制可以通过基因工程改造到逆转录病毒构建体中的程度，并测试我们的诱导系统调节 cDNA 序列受控表达的有效性，从而调节多能 HSC 的生长。我们的首要目标是开发和评估表达报告基因（荧光素酶和 GFP）以及 HoxB4 的正交启动子的有效性，HoxB4 已被明确证明在 HSC 的扩增中非常重要。我们的第二个目标是测试用我们的诱导型载体转导的 HSC 中 HoxB4 的调节表达，以强制 HSC 在体内扩增。