TALE的靶向基因操作.pptxVIP

Genecopoeia Inc. 广州复能基因有限公司 4006 020 200 李华平 ;报告提纲;基因功能的确证;Gene trap: 化学诱变、转座子-------随机性的海量筛选 自杀质粒，同源重组：效率低（~1 HR/106 cells），操作难 RNAi：不完全敲除、临时性 表达克隆瞬时转染：操作简便、临时性 病毒介导的整合：整合位点不特异、潜在风险高 ZFN：难以完全找到匹配的3连子锌指、易Off-target TALEN：识别任意目标序列、效率高-----基因操作技术的新里程碑;TALE （transcription activator like effector）;RVD;激活基因的转录-------TALE-TF 特定序列进行切割----TALEN 抑制基因表达 ----TALE-TR 启动子附近的甲基化 --- TALEM 特定序列的重组酶 ---TALER 。。。。。。。 ;TAL-TFs 激活任意目标基因的转录激活因子;TAL-TFs 激活转录示例;TALENs 识别任意特定核酸靶序列的重组核酸酶;DSB;;运用TALEN进行基因修饰的特点和优势;TALEN技术需要注意的问题;Genecopoeia多层次的TALEN功能验证;250;In vitro target DNA cleavage by EGFP-TALENs. (A) The TALEN target plasmid (6110 bp) contains an unique EcoRI site and an eGFP TALEN target site. The two sites are1066 bp apart. (B) 1 μg of the plasmid was incubated with the indicated enzymes for 30 min at 37°C. 0.5 volume of the digestion reaction was analyzed by the agarose gel electrophoresis. * The indicated fragment was checked by PCR, data not shown.;EX-EGFP-Lv105 (200 ng) + Control Plasmid (800 ng) EX-EGFP-Lv105 (200 ng) + EGFP TALENs (800 ng);;Validation of TALENs via HDR Assay;;通过TALEN介导的基因敲除(knock-out)，该基因功能消失或下降;Safe Harbor是指基因组上转录活跃的安全区域，该区域能有效地转录导入的外源基因 同时打断该区域也不会影响细胞的表型，克服了随机整合中常见的转基因沉默、癌变和插入突变等问题。 AAVS1是确证的、广泛使用的、位于人类19号染色体上的Safe Harbor;Human genome safe harbor AAVS1 gene targeting (B) AAVS1 donor control plasmid pAAVS1D-RFP-copGFPpuro (800 ng) was co-transfected with AAVS1 TALEN pair(600 ng for each) or control TALEN pair into HEK293T cells in a 6-well pate. (A) 48 hr post-transfection, the cells were splited 1:10 into a new 6-well pate and selected against 1.0 μg/ml of puromycin. The images were taken after two weeks of selection. The cells with the transfection of pAAVS1D-RFP-copGFPpuro + control TALEN were completely killed by puromycin selection (data no shown). (C) (D) PCR primers designed to detect the HR junction were used to validate the successful integration. ;Components;One of the biggest roadblocks to the application of programmable nucleases is the lac