Development of a Novel Mouse Model to Evaluate HTLV Tax Transformation

描述（由申请人提供）：本课题的目标是建立人类T细胞白血病病毒I型（HTLV-1） Tax癌蛋白在T细胞中有条件表达的新型小鼠转化模型，并利用该模型确定野生型和突变型Tax蛋白的转化活性。HTLV-1是一种人类逆转录病毒，目前全世界约有1500万人感染，是侵袭性恶性肿瘤成人T细胞白血病/淋巴瘤的病因。尽管Tax致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，但其转化细胞的机制尚不清楚。大量的Tax突变体已经产生，它们的生物活性主要在细胞培养系统中得到了彻底的表征。目前该领域面临的一个主要问题是，由于转基因整合位点随机、转基因拷贝数可变和转基因表达水平不一致，无法使用现有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税收基因的载体研究税收转化，这些基因被先前的固定停止盒沉默。这些载体将通过重组被敲入受体小鼠的Rosa26位点。通过将这些小鼠与Lck- cre小鼠杂交，将在发育中的胸腺细胞中特异性切除停止盒，其中Lck启动子是活跃的，允许野生型或突变型Tax蛋白在T细胞中有条件地表达。

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.