Transgenic Organ Cultures

描述（由申请人提供）：在过去的10年里，我们对肾脏发育的分子基础的了解急剧扩大。这些新信息大部分来自于对转基因小鼠和基因靶向小鼠的研究。 然而，这种实验方法存在严重的局限性。首先，在生产每种基因敲除小鼠的过程中涉及成本和时间。其次，以我们自己的Wt 1基因研究为例，后肾原基的早期死亡几乎没有留下任何实际材料来研究，以进一步了解Wt 1参与早期肾脏发育的机制[3，4]。 在组织培养环境中对Wt 1基因的进一步研究对其在早期肾脏发育中的作用的理解几乎没有增加[5]。由于这些局限性，我们一直在寻求开发替代和成本较低的方法来研究早期肾脏发育的分子基础。最近，一些研究已经证明，可以使用电穿孔来实现基因转移到胚胎组织中;这在神经管和肢体发育的研究中特别有用[6-10]。在我们的实验室中，我们正在采用电穿孔技术来实现基因转移到后肾器官培养中。与使用突变小鼠品系相比，有几个优点是显而易见的。(1)在研究特定基因对肾发育的影响时，可以有高得多的通量;（2）使用野生型器官培养物，因此每个胚胎提供2个器官培养物，而不是来自可能是突变纯合的一窝的25%的胚胎;（3）在Wt 1这样的敲除导致细胞凋亡的情况下，可以使用替代实验方法，例如检查功能表型获得的方法。显微注射和电穿孔将用于阐明WT 1在早期肾脏发育中的作用。将通过在后肾间充质中异位表达或抑制Wt 1，并通过原位杂交检查潜在靶基因的表达来测试与Wt 1是否调节各种生长因子相关的假设。

DESCRIPTION (provided by applicant): Our knowledge of the molecular basis for kidney development has expanded dramatically over the last 10 years. Much of this new information has come from the study of transgenic and gene-targeted mice. However, there are serious limitations to this experimental approach. First, there is the cost and time involved in the production of each strain of knockout mouse. Secondly, taking an example from our own work with the Wtl gene, the early demise of the metanephric rudiment leaves little actual material to study in order to gain further insight as to the mechanism by which Wt1 is involved in early kidney development [3, 4]. Further study of the Wt1 gene in tissue culture settings has added little to our understanding of its role in early kidney development [5]. Because of these limitations, we have sought to develop alternative and less costly ways of studying the molecular basis of early kidney development. Recently, several studies have demonstrated that it is possible to use electroporation to achieve gene transfer into embryonic tissues; this has found particular use in the study of neural tube and limb development [6-10]. In our laboratory, we are adapting electroporation technology to achieve gene transfer into metanephric kidney organ cultures. In contrast to work with strains of mutant mice, several advantages are immediately apparent. (1) there can be much higher throughput in studying the effects of specific genes on kidney development; (2) wild type organ cultures are used, hence every embryo provides 2 organ cultures, instead of the 25 % of embryos from a litter that might be homozygous for a mutation; (3) in cases like Wt1, where the knockout causes apoptosis, alternate experimental approaches are available, for example those that examine gain of function phenotypes. Microinjection and electroporation will be used to elucidate the role of WT1 in early kidney development. Hypotheses relating to whether Wtl regulates various growth factors will be tested by ectopically expressing or inhibiting Wt1 in the metanephric mesenchyme, and examining the expression of potential target genes by in situ hyridization.