Novel tumor suppressor gene discovery in pancreatic cancer

描述（由申请人提供）：众所周知，胰腺癌是致命的。充分了解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，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复制第二个拷贝，这是相对罕见的事件。然而，大多数肿瘤抑制基因需要双等位基因失活。RNA干扰（RNAi）是一种能够破坏细胞中特定种类mRNA的新技术。目前，针对所有已知人类基因的库已上市，如果拥有强大的选择系统（例如肿瘤发生），则是基因发现的理想工具。该提案使用该技术来鉴定新的胰腺癌肿瘤抑制基因。假设：新的胰腺癌肿瘤抑制基因可以使用全基因组RNAi文库进行功能鉴定。SA1：构建细胞系。具体地，用全基因组RNAi文库筛选非致瘤性和弱致瘤性胰腺细胞系。SA2：选择致瘤细胞克隆。采用软琼脂生长法和裸鼠肿瘤生长法，筛选具有致瘤性的细胞克隆。SA3：测序RNAi和测试独立RNAi。通过PCR和测序鉴定负责的RNAi，并使用独立的RNAi构建体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.