Generation of Molecularly Defined Inbred Rat Mutants

描述(由申请人提供)：这个项目的长期目标是建立一个冷冻保存的近交系大鼠品系的储存库，每个品系在不同的已知基因中都有精确定义的功能突变缺失。这样的资源将以最低的成本提供给该领域所有有适当资质的研究人员。这个R21应用程序基于我们在鼠标上成功开发的一种新方法。它代表了一个原则证明请求，以便我们将技术转让给RAT，在那里它可能会有更大的用处。我们相信，这种方法将在老鼠身上取得成功，并将R21基金视为一个更大的项目的垫脚石，该项目旨在创建突变老鼠的社区资源。体内将使用一种独特的毛色标记、转座子介导的基因捕获方法来产生突变体。携带睡美人转座子载体pt2/BART3的近交系转基因白化大鼠(F344)将与携带高活性SB转座酶的近交系F344大鼠交配，由已证实的雄性生殖系特异性启动子PGK2：SB11驱动。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。任何有色素的后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微型基因。在制造转基因大鼠时，只需将载体线性化，即可注射。在最初的转基因中，侧翼载体序列起到抑制酪氨酸酶表达的作用。如果转座子被转座酶动员起来在雄性生殖系中跳跃，它就会从这些抑制序列中释放出来，后代变得有色。由于载体在两个方向上都含有剪接受体，基因陷阱使跳跃具有高度的突变性。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通过简并的寡核苷酸聚合酶链式反应从断奶前的尾巴DNA中简单地扩增出来，并进行测序。然后，通过与最近公开的大鼠基因组序列进行比较，可以确定准确的整合点。携带基因陷阱转座子的大鼠跳到基因组中已知或预测的基因或其他感兴趣的区域，可以扩大研究或冷冻保存以供分布。

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.