REVERSIBLE PROMOTER-INSERTION FOR IN VIVO STUDIES OF MELANOMA

描述（由申请人提供）：通常认为，癌症发展的基础过程最好在体内建模。 已经开发了大量的方法用于培养中体细胞中生物过程的遗传解剖;然而，这些技术在体内使用的适应性非常有限。 我们早期的经验表明，可逆的启动子插入诱变提供了许多优势，目前使用的正向遗传学技术培养细胞。 最重要的好处是突变的显性性质，以及容易识别受影响的基因和建立突变和表型之间的因果关系。 将该技术应用于体内研究的主要限制是将插入诱变剂（通常为逆转录病毒载体）递送至所需的细胞类型。 根据我们最近的经验和其他人发表的数据，我们提出了一种方法，通过使用专门设计的转座子载体来规避这一限制。 我们建议应用我们的技术来研究黑色素瘤发展的遗传事件。 我们将筛选在此过程中与致癌Ras（常见于黑色素瘤）合作的遗传事件。 我们将在培养物中确认拟定插入诱变剂的性质，然后继续建立适合诱变的小鼠品系。 在体内验证我们的构建体的性质后，我们将选择性地诱变转基因动物中的黑素细胞，并将测试肿瘤发生和插入的启动子之间的功能联系。 确认的命中将是未来调查的主题，而该技术本身将可用于解剖一般的各种生物现象，特别是肿瘤发生的各个方面。

DESCRIPTION (provided by applicant): It is commonly accepted that the processes underlying the development of cancer are best modeled in vivo. A plethora of methods have been developed for genetic dissection of biological processes in somatic cells in culture; however, the adaptation of these techniques to in vivo use has been very limited. Our earlier experience indicates that reversible promoter-insertion mutagenesis offers a number of advantages over the currently used forward genetics techniques for cultured cells. The most important benefits are the dominant nature of the mutations, and the ease of identifying the affected gene and establishing the causal link between the mutation and the phenotype. The principal limitation for the adaptation of this technique to in vivo studies is the delivery of the insertional mutagen (typically, a retroviral vector) to the desired cell type. Based on our recent experience and published data from others, we propose a method to circumvent this limitation via the use of specially engineered transposon vectors. We propose to apply our technique to study the genetic events underlying melanoma development. We will screen for genetic events that cooperate with oncogenic Ras (commonly found in melanomas) in this process. We will confirm the properties of the proposed insertional mutagens in culture and then proceed to establish mouse strains suitable for mutagenesis. Upon verifying the properties of our constructs in vivo, we will selectively mutagenize melanocytes in transgenic animals and will test the functional link between tumorigenesis and the inserted promoter. Confirmed hits will be the subject of future investigation, while the technique itself will become available for dissection of various biological phenomena in general and aspects of tumorigenesis, in particular.