INDUCIBLE GENE DELETION BY CRE RECOMBINASE TRANSDUCTION

通过 CRE 重组酶转导诱导基因删除

基本信息

批准号：
6629427
负责人：
Pandelakis Andreas Koni
金额：
$ 21.46万
依托单位：
AUGUSTA UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-06-01 至 2005-05-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Genetic engineering in mice has allowed great advances in studies of in vivo biology. A number of technologies and tools have made this possible, from embryonic stem cells and the homologous recombination double-drug selection strategy, to more recent approaches such as the Cre/IoxP system. Cre is a DNA recombinase originally derived from the Escherichia coli (E. coli) P1 bacteriophage that recognizes specific 34 base-pair loxP sites of DNA recombination. The high specificity and efficiency of DNA recombination by Cre in a cofactor-independent manner attracted the attention of the research community as a means to modify the genome of higher organisms. The Cre/loxP system now has a growing number of uses such as conditional gene deletion, transgene activation or inactivation, and translocation between nonhomologous mouse chromosomes. A gene of interest can be deleted in whole animals, for example, by first engineering the gene to strategically incorporate loxP sites. Cre recombinase in the form of a transgene is then employed to mediate deletion of the gene via the loxP sites, perhaps in a cell-type specific or restricted manner. In this way, researchers can, for example, by-pass embryonic lethality that might occur with a conventional knockout mouse approach and/or dissect the in vivo role of a widely expressed gene in specific cell types. The investigators have now generated a novel reagent, TATCre. This protein represents a new approach to DNA recombination, in a temporally controlled fashion, by simply adding TATCre protein to cells. TATCre combines two previously described elements: 1) a peptide sequence identified in the HIV-1 TAT protein that is able to mediate receptor-independent transduction of heterologous proteins into cells (the 'transporter'); and 2) Cre recombinase (the 'cargo'). The investigators intend to develop this new research resource to the stage of being able to induce efficient and complete deletion of loxP-flanked targets in whole mice, by simply injecting mice with TATCre protein. Factors to be considered include the route, dose, and frequency of mouse injection with TATCre, as well as age and sex at the time of injection. The investigators also intend to continue the development of further Cre recombinase-transporter fusion proteins, including forms that employ polyarginine tracts as protein transporters in place of the TAT transporter. Thus, this proposal is intended to allow the development of Cre recombinase protein transduction technology. This approach offers the prospect of achieving induced gene deletion in whole animals and cell lines without the use of a Cre recombinase transgene, and, therefore, provides many more potential avenues for research.

描述（由申请人提供）：小鼠基因工程使体内生物学研究取得了巨大进展。许多技术和工具使这成为可能，从胚胎干细胞和同源重组双药选择策略，到 Cre/IoxP 系统等最新方法。 Cre 是一种 DNA 重组酶，最初源自大肠杆菌 (E. coli) P1 噬菌体，可识别 DNA 重组的特定 34 个碱基对 loxP 位点。 Cre 以不依赖于辅因子的方式进行 DNA 重组，其高特异性和高效率作为修饰高等生物基因组的手段引起了研究界的关注。 Cre/loxP 系统现在的用途越来越多，例如条件基因删除、转基因激活或失活以及非同源小鼠染色体之间的易位。可以在整个动物中删除感兴趣的基因，例如，首先对基因进行工程改造以战略性地整合loxP位点。然后采用转基因形式的 Cre 重组酶通过 loxP 位点介导基因删除，可能以细胞类型特异性或限制性方式。通过这种方式，研究人员可以绕过传统敲除小鼠方法可能发生的胚胎致死性和/或剖析特定细胞类型中广泛表达的基因的体内作用。研究人员现已研制出一种新型试剂 TATCre。这种蛋白质代表了一种新的 DNA 重组方法，只需将 TATCre 蛋白质添加到细胞中，即可以时间控制的方式进行重组。 TATCre 结合了之前描述的两个要素：1）在 HIV-1 TAT 蛋白中鉴定出的肽序列，能够介导异源蛋白不依赖于受体转导至细胞中（“转运蛋白”）； 2) Cre 重组酶（“货物”）。研究人员打算将这种新的研究资源开发到能够通过简单地向小鼠注射 TATCre 蛋白来诱导整只小鼠中 loxP 侧翼靶标有效且完全删除的阶段。需要考虑的因素包括小鼠注射 TATCre 的途径、剂量和频率，以及注射时的年龄和性别。研究人员还打算继续开发更多的 Cre 重组酶-转运蛋白融合蛋白，包括采用聚精氨酸束作为蛋白转运蛋白代替 TAT 转运蛋白的形式。因此，该提案旨在开发 Cre 重组酶蛋白转导技术。这种方法提供了在不使用 Cre 重组酶转基因的情况下在整个动物和细胞系中实现诱导基因删除的前景，因此，提供了更多潜在的研究途径。