课件：慢病毒颗粒制备和应用.ppt

来自GeneCopoeia的慢病毒颗粒制备和应用,GeneCopoeiaTM 4006 020 200 唐珍洁 ,GeneCopoeia Inc.,日本Riken： 2004年1月-4月，为Riken提供1600个人类基因克隆构建。 Roche： 2006年4月-2007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：2006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（美国国家卫生研究院）：2007年7月-2008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2008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。,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 reprogramming,Vierbuchen T, et al.(2010) Direct conversion of fibroblasts to functional neurons by defined factors. Nature 463:1035-1041 Lijian H, et al. (2011) Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors. Nature 475:386-389 Edward E. M, et al. (2011) Highly Efficient miRNA-Mediated Reprogramming of Mouse and Human Somatic Cells to Pluripotency. Cell stem cell 8:376-388,Protocol of Directed Reprogramming,Profile Detection-qPCR Array,Profile 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 /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 /product/search/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,Expression of reprogramming factors -miRNA clone,Pre-made lenti-vector expression clone,Gene Groups/Families,Reserch of reprogramming mechanism,Reserch of reprogramming mechanism,Virus vector,HIV Viron,HIV & replication,Packaging mix -four plasmids system,Mammalian Constitutive promoters,Packaging system,P,Lenti particle Packaging,Packaging Transfection 48 hours,Titration of the Lentiviruses,Florescent 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 unit,Cells for Lentivirus titration : Hela, HT1080, or H1299 cells,Titration of the Lentivirus in 24-well plates,Titration of the Lentiviruses,Florescent 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 unit,Cells for Lentivirus titration : Hela, HT1080, or H1299 cells,Purification and Concentration of Lentiviruses,1. Traditional CsCl ultracentrifugation.,3. Ion exchange: ViraBind Concentration 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 is required High 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.),Figure. Concentration of Lentiviruses: 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).,Application of Lentiviruses,Products and Services offered by GeneCopoeiaTM,谢谢观赏,谢谢观赏,后面内容直接删除就行 资料可以编辑修改使用 资料可以编辑修改使用 资料仅供参考，实际情况实际分析,主要经营：课件设计，文档制作，网络软件设计、图文设计制作、发布广告等 秉着以优质的服务对待每一位客户，做到让客户满意！ 致力于数据挖掘，合