Transgenic Organ Cultures

描述(由申请者提供)：在过去的10年里，我们对肾脏发育的分子基础的知识有了极大的扩展。这些新信息大部分来自对转基因和基因靶向小鼠的研究。然而，这种实验方法有严重的局限性。首先，生产每个品系的基因敲除小鼠所涉及的成本和时间。其次，以我们自己对WTL基因的研究为例，后肾雏形的早期死亡几乎没有留下可供研究的实际材料，以便进一步了解Wt1参与早期肾脏发育的机制[3，4]。在组织培养环境中对Wt1基因的进一步研究几乎没有增加我们对其在早期肾脏发育中的作用的了解[5]。由于这些局限性，我们试图开发替代的、成本更低的方法来研究早期肾脏发育的分子基础。最近，几项研究表明，有可能使用电穿孔来实现基因转移到胚胎组织；这在神经管和肢体发育的研究中发现了特别的用途[6-10]。在我们的实验室，我们正在采用电穿孔技术来实现后肾器官培养中的基因转移。与研究突变小鼠品系相比，几个优势立竿见影。(1)在研究特定基因对肾脏发育的影响方面，可以有更高的吞吐量；(2)使用野生型器官培养，因此每个胚胎提供2个器官培养，而不是来自一窝可能是纯合突变的胚胎的25%；(3)在像Wt1这样的情况下，如果基因敲除导致细胞凋亡，则有其他实验方法可用，例如那些检查功能表型获得的方法。显微注射和电穿孔将被用来阐明WT1在早期肾脏发育中的作用。关于WTL是否调节各种生长因子的假说将通过在后肾间充质中异位表达或抑制Wt1，并通过原位杂交检测潜在靶基因的表达来检验。

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.