慢病毒颗粒制备和应用0524教学课件

1、来自GeneCopoeia的慢病毒颗粒制备和应用GeneCopoeiaTMgenecopoeia 4006 020 200唐珍洁zhenjietangfulengenGeneCopoeia Inc. 日本Riken： 2019年1月-4月，为Riken提供1600个人类基因克隆构建。Roche： 2019年4月-2019年3月，合作完成13000个人类基因克隆的无细胞体系蛋白表达。NCBI ： 2009年成为NCBI推荐克隆供应商。中国科学院广州生物医药与健康研究院：2019年5月至今，慢病毒载体和哺乳动物载体干细胞相关基因表达克隆构建，华南地区特种疾病相关胚胎干细胞库的建立。美国NIH（美国

2、国家卫生研究院）：2019年7月-2019年1月，3000个斑马鱼克隆构建。美国Novartis（美国诺华制药）：2009年10月，美国诺华制药签约长期基因克隆供应商。生命奥秘电子杂志2019年创刊，现已出版45期，拥有近30万名读者。一直关注最前沿资讯，关注内容包括疾病疗法、基因、蛋白、前沿技术等。诱导性多能干细胞肿瘤转移RNAimicroRNA&癌症染色质与干细胞分化p53细胞信号通路GFP基因调控人类基因组测序40年抗癌历程回顾蛋白动力学表观基因组蛋白质泛素化人类蛋白质相互作用生物标志物新型蛋白标签数量遗传学疫苗安全与研发展望新型疫苗载体睡眠机制丙型肝炎抗肿瘤血管生成疗法蛋白激酶与心血管

3、疾病HIV个性化用药人类基因疗法新一代DNA测序技术iPS技术光遗传学技术海量生物学生物信息学在癌症研究中的应用核酸研究生物数据库进展合成生物学转化医学组织工程与再生医学Outline体细胞定向重编程的研究思路慢病毒的构建原理慢病毒颗粒制备方法及应用Application of lentivirus in somatic reprogrammingVierbuchen T, et al.(2019) Direct conversion of fibroblasts to functional neurons by defined factors. Nature 463:1035-1041Lij

4、ian H, et al. (2019) Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors. Nature 475:386-389Edward E. M, et al. (2019) Highly Efficient miRNA-Mediated Reprogramming of Mouse and Human Somatic Cells to Pluripotency. Cell stem cell 8:376-388 Protocol of Directed Rep

5、rogramming重编程因子的选择靶细胞的选择体细胞定向重编程重编程机制研究信号通路探讨终末细胞的筛选和鉴定+检测重编程效率终末细胞的功能指标检测Profile Detection-qPCR ArrayProfile Detection-miRNA qPCR Array包含有65个miRNAs,可用于检测干细胞的分化程度,同时也可筛选特异miRNAs用于体细胞重编程研究 fulengen/product/qpcr/mirna_array/miRNA qPCR Validated Primers人、小鼠、大鼠miRNA的Q-PCR引物都通过严格的实验验证fulengen/product/sea

6、rch/index.php?prt=22输入miRNA name、Acc No或Sequence等即可获得验证结果Expression of reprogramming factors -ORF expression clone基因的ORF表达克隆有6种表达体系，100多种载体；该克隆可直接用于表达miRNA表达框已完全测序确证 基于慢病毒载体系统的表达克隆可转染未分化细胞和难转化的细胞，获得与普通分化细胞类似的转染效率 通过应用eGFP可以监测病毒和质粒载体的转染效率Expression of reprogramming factors -miRNA clonePre-made lenti-

7、vector expression clone已经将约20,000条基因插入到慢病毒载体（Lv105）、哺乳动物载体（M02)等4套载体中，5个工作日即可交付使用。 fulengen/product/clone/orf_clone_special_collection.php Gene Groups/Familiesfulengen/product/search/group/ Reserch of reprogramming mechanism理解细胞正常的分化机制，可以更准确地运用各种重编程因子进行细胞转分化的操作。 fulengen/product/search/pathway/ Rese

8、rch of reprogramming mechanismVirus vectorAdenovirusAAVLentivirusRetrovirusGene ExpressionTransientTransient or StableTransient or StableStableInfect Dividing CellsYesYesYesYesInfect Non-Dividing CellsYesYesYesNoIntegration into Target Cell GenomeNoNo*YesYesImmune Response in Target CellsHighVery Lo

9、wLowModerateRelative Viral TiterXXXXXXXXXXXXRelative Transduction EfficiencyXXXXXXXXXXXX*Native AAV will integrate, but recombinant AAV rarely does.HIV Viron慢病毒载体（Lentiviral Vector ）主要是以HIV-1（人类免疫缺陷I型病毒）为基础发展起来的基因转移载体 。HIV & replicationPackaging mix -four plasmids systemCMVgagpolRREpoly AEF1REVpoly

10、ACMVpoly AVSV-G包膜蛋白质粒包装质粒+Mammalian Constitutive promotersQin JY, et al. (2019) Systematic Comparison of Constitutive Promoters and the Doxycycline-Inducible Promoter. PLoS ONE 5(5): e10611Packaging systemPLenti particle PackagingPlate 293T cells for transfection.Prepare plasmid mix + EndoFectinAdd

11、to 293T cellsChange MediumHarvest medium (Viruses)Lentivirus titrationLentivirus purification and concentration. (optional)Store Lentiviruses in single use aliquot at -80CDay 1Day 2Day 4Day 4-724 - 48hours20-30min4-6 h48-72hours after transfectionPackaging Transfection 48 hours1-100ms-48h2-100ms-48h

12、3-100ms-48hTitration of the LentivirusesFlorescent colony counting or FACS counting: Infection units (IU/ml)Resistant colony counting: Infection units (IU/ml)RT-qPCR: copy number of the virus, not the infection unitCells for Lentivirus titration : Hela, HT1080, or H1299 cellsTitration of the Lentivi

13、rus in 24-well plates1- 2.5 x 107 U/ml 2- 1.8 x 107 U/ml 3- 3.5 x 107 U/ml 0.1ul1- 2.5 x 107 U/ml 1.0 ul2- 1.8 x 107 U/ml 3- 3.5 x 107 U/ml 将慢病毒颗粒稀释 成不同浓度(0.02, 0.1, 1.0 and 5.0 ul)转染细胞4872h后，在荧光显微镜下计数滴度=感染细胞数*病毒颗粒剂量Titration of the LentivirusesFlorescent colony counting or FACS counting: Infection

14、units (IU/ml)Resistant colony counting: Infection units (IU/ml)RT-qPCR: copy number of the virus, not the infection unitCells for Lentivirus titration : Hela, HT1080, or H1299 cellsPurification and Concentration of Lentiviruses1. Traditional CsCl ultracentrifugation.3. Ion exchange: ViraBind Concent

15、ration & Purification Kits (Cell Biolabs) ; Fast-Trap Virus Purification and Concentration Kits (Millipore).4. Precipitation: LentiPacTM Lentivirus Concentrator(GeneCopoeia).Quick and simple concentration of lentiviral particles(2-3 hours).Increase titer (IU/ml) by 10X 100X. No ultracentrifugation i

16、s requiredHigh recovery : 90%Pre-step: Filtration (polyethersulfone (PES) filters) or general centrifugation. 2. Ultra filtration: using filter 0.01 M (200KD) Purification and Concentration of Lentiviruses (Cont.)A. Pre 0.2ulB. Conc. 4h 0.02ulC. Conc. 16h 0.02ulFigure. Concentration of Lentiviruses:

17、 GFP containing lentiviruses (2x107IU/ml) were concentrated 10 times using LentiDens and then transduced the H1299 cells in 24-well plates. The images were taken 48 hours after the transduction. A: 0.2 ul of pre-concetrated lentiviruse; B: 0.02ul of concentrated lentivirus (incubated 4 hours with LentiDens before centrifuge). C: 0.02ul of concentrated lentivirus (incubated 16 hours with LentiDens before centrifuge). Ap