Novel tumor suppressor gene discovery in pancreatic cancer

描述(申请人提供)：胰腺癌是出了名的致命疾病。充分了解在这种癌症中突变的癌基因和肿瘤抑制基因将有助于诊断和治疗。直到最近，在二倍体细胞中进行遗传筛选一直很困难。尽管存在强大的选择系统，但一个等位基因的插入突变只能通过产生显性负基因产物来灭活第二个副本，这是一种相对罕见的事件。然而，大多数肿瘤抑制基因需要双等位基因失活。RNA干扰(RNAi)是一种可以破坏细胞中特定种类的mRNA的新技术。现在，针对所有已知人类基因的文库已经商业化，如果拥有强大的选择系统，例如肿瘤发生学，它是发现基因的理想工具。这项建议使用这项技术来识别新的胰腺癌抑癌基因。假设：利用全基因组RNAi文库可以从功能上鉴定新的胰腺癌抑癌基因。SA1：构建细胞系。具体地说，利用全基因组RNAi文库转导非致癌和弱致癌的胰腺细胞系。SA2：选择致瘤细胞克隆。利用软琼脂中的生长和裸鼠体内的肿瘤生长，选择已成为致瘤细胞的克隆。SA3：对RNAi进行测序并测试独立的RNAi。通过聚合酶链式反应和测序确定负责的RNAi，并使用独立的RNAi构建来确认可疑的肿瘤抑制基因。

DESCRIPTION (provided by applicant): Pancreatic cancer is notoriously lethal. A full understanding of the oncogenes and tumor suppressor genes mutated in this cancer will aide both diagnostically and therapeutically. Genetic screens in diploid cells have been difficult until recently. Despite the existence of powerful selection systems, insertional mutagenesis of one allele can only inactivate the second copy by producing a dominant negative gene product, a relatively rare event. Most tumor suppressor genes however require bi-allelic inactivation. RNA interference (RNAi) is a novel technology that can destroy a given species of mRNA in cells. Libraries are now commercially available that target all known human genes, and are ideal tools for gene discovery if one has a powerful selection system, such as tumorigenesis. This proposal uses this technology to identify novel pancreatic cancer tumor suppressor genes. Hypothesis: Novel pancreas cancer tumor suppressor genes can be functionally identified using whole genome RNAi libraries. SA1: Construct cell lines. Specifically, transduce non-tumorigenic and weakly- tumorigenic pancreas cell lines with whole-genome RNAi libraries. SA2: Select tumorigenic cell clones. Using growth in soft agar and tumor growth in nude mice, and select cell clones that have become tumorigenic. SA3: Sequence RNAi's and test independent RNAi's. Identify the responsible RNAi by PCR and sequencing, and confirm suspect tumor suppressor genes using independent RNAi constructs.