ISSA: novel functional approach to cancer-related genes

描述（由申请人提供）：鉴定有助于肿瘤发生的基因和途径应导致确定治疗干预的新靶点，并为个体癌症患者提供更好的诊断，分期和风险评估的生物标志物。基于基因表达调节引起的表型变化来分配基因功能的可靠方法将极大地促进癌症分子遗传学的进一步发展。现有的这种技术是基于筛选基因修饰细胞的遗传因素，有利于细胞在限制性条件下生长（积极选择）。最近，一种新的基因发现方法被开发出来，称为选择减法（SSA），允许直接的负选择。尽管SSA已被证明可用于分离杀伤或生长抑制遗传元件，但其能力受到构建表达库的必要性的限制。这一建议的重点是开发一种新的插入选择减法方法（ISSA），该方法结合了SSA的优势和逆转录病毒插入突变的力量，并基于一个全新的载体系统。插入诱变臂通过添加可调节的启动子、剪接供体序列和捕获聚腺苷化信号的能力而增强。ISSA的第二部分包括“标记”或“条形码”每个突变体（SSA），从而允许在选择过程中使用“减噬阵列”（SSA的关键组成部分）来监测种群中突变体的相对丰度。ISSA技术有望成为一种通用的功能筛选工具，摆脱其前身的主要缺点。在对ISSA进行“技术”测试后，其功效将由参与细胞对肿瘤坏死因子敏感性调节的基因鉴定来确定，肿瘤坏死因子是一个已经通过多种方法（包括功能选择）进行了很好研究的具有良好特征的系统。

DESCRIPTION (provided by applicant): Identification of genes and pathways that contribute to tumorigenesis should lead to the defining of novel targets for therapeutic intervention and provide biomarkers for better diagnosis, staging, and risk assessment for individual cancer patients. Further progress in molecular genetics of cancer would greatly benefit from a reliable methodology of assigning gene functions based on phenotypic changes resulting from modulations in gene expression. Existing techniques of this kind are based on screening of genetically modified cells for genetic elements favoring cell growth under restrictive conditions (positive selection). Recently, a novel gene discovery methodology, named selection subtraction approach (SSA), was developed that allowed for a direct negative selection. Although proven useful for isolation of killing or growth suppressive genetic elements, SSA's capabilities are limited by the necessity to construct expression libraries. This proposal is focused on developing a new Insertional Selection Subtraction Approach (ISSA) that combines the advantages of SSA with the power of retroviral insertional mutagenesis and is based on a completely new vector system. The insertional mutagenesis arm is enhanced by the addition of a regulatable promoter, splice donor sequences, and the ability to trap polyadenylation signals. The second arm of ISSA involves "tagging" or "bar-coding" each mutant (SSA), thereby allowing to monitor the relative abundance of mutants within the population during selection by using "retrophage arrays," the key component of SSA. ISSA technique promises to become a universal functional screening tool, free from major drawbacks of its precursors. After "technical" testing of ISSA, its power will be determined by identification of genes involved in regulation of cell sensitivity to TNF, a well-characterized system that has been already well studied by numerous approaches, including functional selection.