IN VIVO GENE TARGETING AND GENE THERAPY IN ZEBRAFISH

到目前为止，脊椎动物的基因靶向只能在小鼠身上实现。 对其他脊椎动物类似突变的比较研究将 允许进行有助于确定重要性的更完整的分析 与人类基本过程有关的保守基因 发展和疾病。因为斑马鱼的胚胎几乎 在开发过程中透明且易于观察，是一种极好的 脊椎动物胚胎发生的模型。然而，高效的转基因 斑马鱼尚未建立，主要是由于缺乏 高水平整合报告基因表达的载体系统 在整个生命周期中都是可屏蔽的。一种非病毒载体pFRMwg具有 设计了稳定表达绿色荧光蛋白(GFP)的基因 几乎无处不在的斑马鱼。此外，转基因 技术已经得到优化，可以对同源基因进行筛选 活胚胎中的重组事件。在拟议的研究中，这一点 将对矢量系统进行修改，以使综合测试仪轨迹 含有绿色荧光蛋白蓝色突变的人可以通过显微注射 绿色绿色荧光蛋白载体的1-细胞胚胎及绿色荧光蛋白筛选 在发育过程中的表达。该基因在体内的频率 目标定位将作为载体设计的变量进行测试和优化 并对选择进行显微注射的胚胎类型进行操作。这个 目标将是为高效的生殖系创造条件 基因靶向事件的转移。 人类基因疗法为治愈和治疗提供了巨大的潜力 预防疾病。再一次，主要的动物模型是 鼠标，在这个系统中有几个明显的限制： 基因治疗载体的表达在活体中不易筛选 小鼠，而且意外的生殖系转移的可能性只能是 通过雄配子的聚合酶链式反应进行大规模检测。此外，从长远来看 表达的持久性(例如1年)尚未显示。使用 PFRMwg载体作为斑马鱼基因治疗的标记，表达可以 在活鱼中得分。在本研究中，坚持与本土化 将作为DNA变量的函数进行测试 剂量、DNA同化辅助因子和给药途径 与载体DNA随时间的状态相关。我们的目标是 实现持久的高水平表达，并最终估计 在特定处理后的生殖系转移的可能性。

Vertebrate gene targeting has so far been possible only in the mouse. Comparative studies of similar mutations in other vertebrates would allow a more complete analysis useful for establishing the importance of conserved genes implicated in fundamental processes of human development and disease. Because the zebrafish embryo is nearly transparent and easily observed during development, it is an excellent model of vertebrate embryogenesis. However, efficient transgenesis of the zebrafish has not yet been established primarily due to the lack of vector systems capable of high level integrated reporter gene expression screenable throughout the life cycle. A non viral vector, pFRMwg, has been designed which stably expresses the green fluorescent protein (GFP) almost ubiquitously throughout the zebrafish. In addition, transgenic techniques have been optimized to allow screening of homologous recombination events in the living embryo. In the proposed study, this vector system will be modified so that an integrated tester locus containing a blue mutation of GFP can be targeted by microinjection of the 1-cell embryo with green GFP vectors and screened for green GFP expression during development. The frequency of this in vivo gene targeting will be tested and optimized as the variables of vector design and the type of embryos chosen to be microinjected are manipulated. The goal will be to establish the conditions for efficient germ line transfer of gene targeting events. Human gene therapy offers a tremendous potential for both the cure and prevention of disease. Again, the primary animal model has been the mouse, and several limitations have become apparent in this system: expression of gene therapy vectors can not be easily screened in live mice, and the potential of unintended germ line transfer can only be tested on a large scale by PCR of male gametes. Additionally, long term persistence of expression (e.g. 1 year) is yet to be shown. Using the pFRMwg vector as a marker for gene therapy in zebrafish, expression can be scored in living fish. In this study, persistence and localization of this expression will be tested as a function of the variables of DNA dose, DNA assimilation cofactors and route of administration and finally correlated with the state of the vector DNA over time. The goals are to achieve persistent high level expression and eventually to estimate the potential of germ line transfer after a particular treatment.