Electroporation system for zebrafish development and genetics

我们拟建立一套活体电穿孔系统，用于研究成年斑马鱼鳍再生的分子机制，以及研究斑马鱼脑的发育和功能。斑马鱼是了解脊椎动物生物学和发育的重要模型系统，胚胎、幼虫和成体组织的可及性使其特别适合通过电穿孔进行基因操作研究。发育生物学和分子遗传学的一个重要目标是确定基因的功能。 这可以通过检查给定基因的表达丧失（功能丧失）或相反，其在通常不表达的结构域中的表达（功能获得）对动物的正常发育或器官功能的影响在体内进行。 体内电穿孔是近年来发展起来的一种将基因转移到胚胎和成年动物特定组织的方法。 在该方法中，首先将带电分子显微注射到感兴趣的组织/器官中。 然后，电脉冲被传递到组织，以在细胞膜中产生允许注射分子进入的孔的瞬时形成。 当应用于胚胎和成年动物时，该技术可以将DNA或其他带电大分子有效引入整个器官的细胞或仅少数细胞中，具体取决于所使用的电极类型。 因此，它不一定会损害胚胎的发育或成年人的生活。 这种基因递送方法具有快速、简便和成本效益高的优点。 在斑马鱼中，它提供了允许在动物生命的所有阶段进行基因操作研究的额外优势，而在受精卵中单独进行显微注射通常会限制引入的大分子的作用持续时间。

We propose to acquire an in vivo electroporation system to investigate the molecular mechanisms underlying fin regeneration in the adult zebrafish and to study zebrafish brain development and function. The zebrafish is an important model system to understand vertebrate biology and development and the accessibility of embryos, larvae, and adult tissue make it particularly amenable for gene manipulation studies by electroporation. An important goal in developmental biology and molecular genetics is to determine the function of genes.  This can be done in vivo by examining the consequences of the loss of the expression of a given gene (loss of function) or, on the contrary, its expression in a domain where it is normally not expressed (gain of function), on the normal development of the animal or the function of an organ.  In vivo electroporation is a recently developed gene transfer method to specific tissues of embryos and adult animals.  In this method, charged molecules are first microinjected in the tissue/organ of interest.  Then electric pulses are delivered to the tissue to generate the transient formation of pores in the cells membranes that allow the entry of the injected molecules.  When applied to embryos and adult animals, this technique allows the efficient introduction of DNA or other charged macromolecules into cells of an entire organ or of only a few cells, depending on the type of electrodes used.  Therefore, it does not necessarily compromise the development of the embryo or the life of the adult.  This method of gene delivery presents the advantages of being rapid, easy and cost-effective.  In zebrafish, it offers the additional advantage of allowing gene manipulation studies at all stages of the life of the animal, in contrast to microinjection alone performed in fertilized eggs which often limits the duration of the effect of the introduced macromolecule.