Generation of Molecularly Defined Inbred Rat Mutants

分子定义的近交大鼠突变体的产生

• 批准号: 7140309
• 负责人: COLIN Edward BISHOP
• 金额: $ 0.17万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7140309
• 关键词: Generation; Molecularly; Defined; Inbred; Rat

DESCRIPTION (provided by applicant): The long-term goal of this project is to produce a cryopreserved repository of inbred rat strains each having a precisely defined loss of function mutation in a different known gene. Such a resource will be made available to all suitably qualified researchers in the field for minimal cost. This R21 application is based on a novel approach we have successfully developed in the mouse. It represents a proof-of-principle request in order for us to transfer the technology to the rat where it may be of even greater utility. We are confident that this approach will be successful in the rat and see this R21 funding as a stepping-stone to a larger project designed to create a community resource of mutant rats. Mutants will be produced in vivo using a unique coat color tagged, transposon mediated, gene trapping approach. An inbred transgenic albino rat (F344) carrying the Sleeping Beauty (SB) transposon vector, pT2/BART3 will be mated to an inbred F344 rat carrying a hyperactive SB transposase, driven by the proven male germ line specific promoter, PGK2: SB11. Male F1 rats carrying both transgenes will then be mated to normal albino F344 females. Any progeny that are pigmented will represent transposon jumps. The pigmentation occurs because the transposon vector, pT2/BART3 contains the tyrosinase minigene which is able to rescue albinism in rodents. When making the transgenic rats the vector is simply linearized for injection. The flanking vector sequences act to inhibit tyrosinase expression in the initial transgenic. If the transposon is mobilized to jump in the male germ line by the transposase, it is released from these inhibitory sequences, and the progeny becomes pigmented. As the vector also contains splice acceptors in both orientations, gene trapping makes jumps highly mutagenic. In our mouse model the jumping frequency is currently 50-60%. The great power of this approach is that the insertion points of the transposon jumps can be simply amplified from pre-weaning tail snip DNA, using degenerate oligo PCR, and sequenced. The precise integration point can then be identified by comparison with the recently available public rat genome sequence. Rats with gene trap transposon jumps into known or predicted genes, or other areas of interest, in the genome can be expanded for study or cryopreserved for distribution.

描述（由申请人提供）：该项目的长期目标是建立一个冷冻保存库，保存近亲繁殖的大鼠菌株，每个菌株在不同的已知基因中具有精确定义的功能丧失突变。这种资源将以最低的费用提供给该领域所有适当合格的研究人员。这个R21应用程序是基于我们在鼠标中成功开发的一种新方法。它代表了一个原理证明请求，以便我们将技术转让给老鼠，在那里它可能会有更大的效用。我们相信这种方法将在大鼠身上取得成功，并将R21基金视为一个更大项目的垫脚石，该项目旨在创建突变大鼠的社区资源。突变体将在体内产生，使用独特的毛色标记，转座子介导，基因捕获方法。一只携带睡美人（SB）转座子载体pT2/BART3的近交转基因白化大鼠（F344）将在已证实的雄性生殖系特异性启动子PGK2: SB11的驱动下，与携带过度活跃SB转座酶的近交F344大鼠交配。携带这两种转基因的雄性F1大鼠将与正常的白化F344雌性大鼠交配。任何着色的子代都代表转座子跳跃。色素沉着的发生是因为转座子载体pT2/BART3含有能够拯救啮齿动物白化病的酪氨酸酶基因。在制造转基因大鼠时，将载体简单地线性化以供注射。在初始转基因中，侧翼载体序列抑制酪氨酸酶的表达。如果转座子被转座酶调动到雄性种系中，它就会从这些抑制序列中释放出来，后代就会着色。由于载体在两个方向上都含有剪接受体，基因捕获使得跳跃具有高度的诱变性。在我们的小鼠模型中，跳跃频率目前为50-60%。这种方法的强大之处在于，转座子跳跃的插入点可以简单地从断奶前的尾巴片段DNA中扩增出来，使用退化寡聚PCR，并测序。然后可以通过与最近公开的大鼠基因组序列的比较来确定精确的整合点。携带基因诱捕转座子的大鼠进入已知或预测的基因，或基因组中其他感兴趣的区域，可以扩展研究或冷冻保存以供分布。