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生成一个冷冻保存的近交大鼠菌株的存储库，每个存储库在一个不同的已知基因中具有精确定义的功能突变丧失。这样的资源将以最低的成本提供给该领域的所有合格的研究人员。该R21应用程序基于我们在鼠标中成功开发的一种新颖方法。它代表了原理证明请求，以便我们将技术转移到可能具有更大效用的老鼠中。我们相信这种方法将在老鼠中取得成功，并将这笔R21资金视为旨在创造突变大鼠社区资源的大型项目的垫脚石。突变体将使用独特的涂层颜色标记，转座子介导的基因诱捕方法在体内产生突变体。带有睡美人（SB）转座子矢量的近交性转基因白化大鼠（F344），PT2/BART3将与携带过度活跃的SB转座酶的近近繁殖F344大鼠配合，由经过验证的男性生殖系特异性启动子PGK2：SB11驱动。然后，携带两种转基因的雄性F1大鼠将与正常的白化病F344雌性配对。任何有色的后代都代表转座子跳跃。出现色素沉着是因为转座子载体PT2/BART3包含能够在啮齿动物中挽救白化病的酪氨酸酶的小蛋白酶。当使转基因大鼠使矢量简单地进行了线性化以进行注射。侧面矢量序列起作用可抑制初始转基因中酪氨酸酶的表达。如果转座子通过转座酶在男性生殖系中跳跃，则从这些抑制性序列中释放出来，后代会变色。由于矢量在两种方向上还包含剪接受体，因此基因捕获使跳跃高度诱变。在我们的鼠标模型中，跳跃频率当前为50-60％。这种方法的强大力量是，可以使用简并寡核PCR并进行测序，可以简单地从预断奶的尾部剪接DNA中放大转座子跳跃的插入点。然后，可以通过与最近可用的公共大鼠基因组序列进行比较来识别精确的集成点。具有基因陷阱转座子的大鼠可以跳入已知或预测的基因或其他感兴趣的区域，可以扩展以进行研究或冷冻保存进行分布。