2016-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/648973摘要：CRISPR/Cas9 技術是一個革命性的基因組編輯工具，可用來研究基因功能。全基因體的CRISPR 庫可用來正向或負向選擇出對各種生物現象具重要性的基因。在本計劃中，我們打算以CRISPR 庫技術在肝癌中進行抑癌基因的功能性篩選。我們把慢病毒CRISPR 庫GeCKO 轉染到肝癌細胞株HepG2 及HA22T，之後把這些細胞打入NOD/SCID 小鼠皮下。我們收集注射前，注射後兩週及注射後六週的檢體。我們以深層定序法決定各個檢體的單一指引RNA (sgRNA)的成分和頻率。之後我們把豐富化的sgRNA 單株化，再轉染到肝癌細胞株。我們將以依賴錨定和不依賴錨定的細胞生長測定及小鼠體內的腫瘤生成測定以評估敲毀目標基因對細胞生長的影響。此外，我們也將以Boyden 室侵犯及遷移測定及傷口癒合測定以評估敲毀目標基因對腫瘤侵犯的影響。最後，依目標基因的特徵，我們將進行功能性研究以找出這些基因抑制癌症的分子機轉。<br> Abstract: CRISPR/Cas9 technology is a revolutionary genome editing tool for study of gene function.Genome-wide CRISPR library can be used for positive and negative selection of genes important forbiological events. In this proposal, we plan to use the CRISPR library technology to perform a functionalscreening of tumor suppressor gene in liver cancer. The lentiviral CRISPR library GeCKO is transducedinto liver cancer cell lines HepG2 and HA22T. The cells are subcutaneously injected into NOD/SCID mice.Samples are collected at before injection, 2 weeks and 6 weeks after injection. The component andfrequency of single guide RNA (sgRNA) in each sample are determined by deep sequencing. The enrichedsgRNAs are cloned and transduced into liver cancer cell lines. Anchorage-dependent andanchorage-independent cell growth assays and mice in vivo tumorigenic assays are used to evaluate theeffects of knockout of target genes on cell proliferation. Boyden chamber invasion and migration assays andthe wound-healing assay are used to evaluate the effects of knockout of target gens on tumor invasion.Finally, depending on the characteristics of the target genes, we will perform functional studies to identifythe molecular mechanisms of tumor suppression of these genes.