Development of a Novel Mouse Model to Evaluate HTLV Tax Transformation

开发新的小鼠模型来评估 HTLV 税收转型

• 批准号: 8488975
• 负责人: SUSAN J MARRIOTT
• 金额: $ 20.03万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8488975
• 关键词: Adult; Apoptosis; Binding; Biological; Biological Models; Biological Process; Blood Cells; C57BL/6 Mouse; CREB1 gene; Cell Culture System; Cell physiology; Cells; Cellular biology; Communities; Development; Disease; Gene Dosage; Gene Expression; Gene Silencing; Genetic Recombination; Goals; Human; Human T-Cell Leukemia Viruses; Human T-lymphotropic virus 1; Infection; Knock-in Mouse; Knowledge; Link; Malignant Neoplasms; Mediating; Modeling; Mus; Mutation; NF-kappa B; Oncogene Proteins; Pathway interactions; Pharmaceutical Preparations; Play; Problem Solving; Protein Family; Proteins; Resources; Retroviridae; Role; Site; T-Cell Leukemia; T-Lymphocyte; Taxes; Time; Transgenes; Transgenic Mice; Transgenic Model; Viral; Virus; Work; cancer type; cell transformation; improved; innovation; insight; leukemia; leukemia/lymphoma; member; metaplastic cell transformation; mouse model; mutant; novel; novel therapeutics; promoter; protein expression; public health relevance; tax Gene Products; tax Genes; thymocyte; tool; transgene expression; tumor; tumorigenesis; tumorigenic; uncontrolled cell growth; vector

DESCRIPTION (provided by applicant): The goal of this proposal is to establish a novel mouse transformation model in which the human T cell leukemia virus type I (HTLV-1) Tax oncoprotein is conditionally expressed in T cells, and to use this model to determine the transforming activity of wild-type and mutant Tax proteins. HTLV-1 is a human retrovirus that currently infects approximately 15 million people in the world and is the cause of the aggressive malignancy, adult T cell leukemia/lymphoma. Although the Tax oncoprotein has been shown to transform cells in culture and to induce tumors in a variety of transgenic mouse models, the mechanism by which Tax transforms cells is not well understood. A large number of Tax mutants have been generated and their biological activities have been thoroughly characterized primarily in cell culture systems. A major problem currently facing the field is that the transforming activity of Tax mutants cannot be compared using available transgenic models due to random transgene integration sites, variable transgene copy number and inconsistent transgene expression levels. Thus, it is very difficult to link the biological activities of Tax mutants with their transforming potential. To solve this problem we will develop an innovative mouse model in which to study Tax transformation using vectors containing wild-type or mutant Tax genes that are silenced by a preceding floxed stop cassette. These vectors will be knocked in to the Rosa26 locus of recipient mice by recombination. By crossing these mice with Lck-CRE mice, the stop cassette will be specifically excised in developing thymocytes where the Lck promoter is active, allowing conditional expression of wild-type or mutant Tax proteins in T cells, the natural target of HTLV-1 infection. Insertion into the Rosa26 locus will eliminate inconsistent integration sites and standardize gene copy number resulting in consistent levels of wild-type and mutant Tax protein expression. The mouse model will be established in Aim 1 by creating targeting vectors containing silenced wild-type or mutant Tax genes. Targeting vectors will be knocked in to the Rosa26 locus of C57BL/6 mice, which will then be crossed with homozygous Lck-CRE mice, thereby excising the stop cassette to generate mice expressing wild-type or mutant Tax specifically in T cells. Aim 2 will examine the effect of mutations that disable specifi biological functions of Tax on Tax- mediated tumorigenesis. Tax can bind to and regulate the activity of members of the SRF, CREB, NF-kB and PBM protein families, each of which has been implicated in oncogenesis. Mice established in Aim 1 will be used to compare for the first time the tumorigenic potential of wild-type and mutant Tax proteins in an effort to identify pathways that are required for Tax tumorigenesis. The proposed studies will establish a new mouse model that will overcome current limitations and provide greater insight into the mechanism of HTLV-1 Tax transformation, knowledge that is currently lacking and that promises to yield novel insights into viral and cellular biology.

描述（由申请人提供）：本提案的目的是建立一种新型小鼠转化模型，其中人T细胞白血病病毒I型（HTLV-1）Tax癌蛋白在T细胞中条件性表达，并使用该模型测定野生型和突变型Tax蛋白的转化活性。HTLV-1是一种人类逆转录病毒，目前感染了世界上约1500万人，并且是侵袭性恶性肿瘤成人T细胞白血病/淋巴瘤的原因。虽然Tax癌蛋白已被证明在培养中转化细胞并在各种转基因小鼠模型中诱导肿瘤，但Tax转化细胞的机制尚不清楚。已经产生了大量的Tax突变体，并且它们的生物活性已经主要在细胞培养系统中被彻底表征。该领域目前面临的主要问题是，由于随机的转基因整合位点、可变的转基因拷贝数和不一致的转基因表达水平，不能使用可用的转基因模型来比较Tax突变体的转化活性。因此，很难将Tax突变体的生物学活性与其转化潜力联系起来。为了解决这个问题，我们将开发一种创新的小鼠模型，其中使用含有野生型或突变型Tax基因的载体来研究Tax转化，所述野生型或突变型Tax基因被前面的floxed终止盒沉默。这些载体将通过重组敲入受体小鼠的Rosa 26基因座。通过将这些小鼠与Lck-CRE小鼠杂交，终止盒将在Lck启动子有活性的发育中的胸腺细胞中被特异性切除，从而允许野生型或突变型Tax蛋白在T细胞中的条件表达， HTLV-1感染的天然靶点。插入Rosa 26基因座将消除不一致的 整合位点和标准化基因拷贝数，导致野生型和突变Tax蛋白表达水平一致。将在目标1中通过创建含有沉默的野生型或突变型Tax基因的靶向载体来建立小鼠模型。将靶向载体敲入C57 BL/6小鼠的Rosa 26基因座，然后将其与纯合Lck-CRE小鼠杂交，从而切除终止盒以产生在T细胞中特异性表达野生型或突变型Tax的小鼠。目的2将检测使Tax的特定生物学功能丧失的突变对Tax介导的肿瘤发生的影响。Tax可以结合并调节SRF、CREB、NF-kB和PBM蛋白家族成员的活性，这些蛋白家族中的每一个都与肿瘤发生有关。Aim 1中建立的小鼠将首次用于比较野生型和突变型Tax蛋白的致瘤潜力，以确定Tax肿瘤发生所需的途径。拟议的研究将建立一种新的小鼠模型，该模型将克服目前的局限性，并对HTLV-1 Tax转化机制提供更深入的了解，这是目前缺乏的知识，有望对病毒和细胞生物学产生新的见解。