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: 7323623
• 负责人: COLIN Edward BISHOP
• 金额: $ 14.47万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 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 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7323623
• 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）：本项目的长期目标是生产近交系大鼠品系的冷冻保存库，每种品系在不同的已知基因中具有精确定义的功能缺失突变。这一资源将以最低的费用提供给该领域所有合格的研究人员。这个R21应用程序是基于我们在小鼠中成功开发的一种新方法。它代表了一个原理证明的要求，以便我们将技术转移到老鼠身上，在那里它可能会有更大的用处。我们相信这种方法将在大鼠中取得成功，并将R21资金视为旨在创建突变大鼠社区资源的更大项目的垫脚石。突变体将使用独特的毛色标记、转座子介导的基因捕获方法在体内产生。携带睡美人（SB）转座子载体pT 2/BART 3的近交转基因白化病大鼠（F344）将与携带由经证实的雄性生殖系特异性启动子PGK 2：SB 11驱动的超活性SB转座酶的近交F344大鼠交配。然后将携带两种转基因的雄性F1大鼠与正常的白化病F344雌性大鼠交配。任何着色的后代将代表转座子跳跃。色素沉着的发生是因为转座子载体pT 2/BART 3含有酪氨酸酶小基因，该基因能够拯救啮齿动物中的白化病。当制备转基因大鼠时，载体被简单地线性化用于注射。侧翼载体序列用于抑制初始转基因中的酪氨酸酶表达。如果转座酶动员转座子在雄性种系中跳跃，它就会从这些抑制序列中释放出来，后代就会变得有色素。由于载体还含有两个方向的剪接受体，基因捕获使得跳跃高度致突变。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使用简并寡聚PCR从断奶前的剪尾DNA中简单地扩增并测序。精确的整合点可以通过与最近公开的大鼠基因组序列进行比较来确定。具有基因陷阱转座子跳跃到基因组中已知或预测的基因或其他感兴趣的区域的大鼠可以扩增用于研究或冷冻保存用于分布。