慢病毒载体稳定转染人角质形成细胞克隆的整合位点倾向性分析及其体外生长特性分析-干细胞工程专业毕业论文.docx

第三军医大学硕士学位论文 第三军医大学硕士学位论文 PAGE PAGE 2 min minute 分钟 ml milliliter 毫升 MLV murine leukemia virus 小鼠白血病病毒 MMTV mouse mammary tumor virus 小鼠乳房肿瘤病毒 MSCV murine stem cell virus 小鼠干细胞病毒 OD optical density 光密度 PBS phosphate buffered saline 磷酸盐缓冲盐溶液 PERV porcine endogenous retrovirus 猪内源性逆转录病毒 PI propidium iodide 碘化丙啶 rpm revolutions per minute 转/分 sec second 秒 SINE short interspersed nuclear element 短散布核元件 SIV simian immunodeficiency virus 猿猴免疫缺陷病毒 TSS transcription start site 转录起始位点 XMRV xenotropic murine leukemia virus- related virus 异嗜性鼠白血病病毒相关病毒 Study on the analysis of integration site preferences in human keratinocyte clones with lentiviral vector stable transduction and their growth characteristics in vitro Abstract Background: Lentiviral vector stably integrates into the host genome, which is likely to result in silencing of transgene expression and insertional mutagenesis due to interactions between the provirus and its host. A genome-wide analysis of lentiviral vector integration site preferences has become an important tool to monitor vector-on-host interactions and evaluate application security of the vector in preclinical animal models and clinical gene therapy trials. Four human keratinocyte clones with lentiviral vector stable transduction were obtained in the previous study in our labarotory and named clone 1, clone 2, clone 3 and clone 4. If the integration site distributions in these four clones are uncovered, it will increase our knowledge of lentiviral vector integration site preferences in the genome of human epidermal cells. In addition, if lentiviral vector inserts into or indirectly affects genes controlling cell growth, development and differentiation, some changes of growth characteristics or malignant transformation will probably happen in the host cell. Therefore, it is also necessary to observe the growth characteristics of the four clones in vitro. In the present study, in order to provide experimental evidences for assessing the global bio