慢病毒顆粒制備和應(yīng)用0524教學課件

1、來自GeneCopoeia的慢病毒顆粒制備和應(yīng)用GeneCopoeiaTMgenecopoeia 4006 020 200唐珍潔zhenjietangfulengenGeneCopoeia Inc. 日本Riken： 2019年1月-4月，為Riken提供1600個人類基因克隆構(gòu)建。Roche： 2019年4月-2019年3月，合作完成13000個人類基因克隆的無細胞體系蛋白表達。NCBI ： 2009年成為NCBI推薦克隆供應(yīng)商。中國科學院廣州生物醫(yī)藥與健康研究院：2019年5月至今，慢病毒載體和哺乳動物載體干細胞相關(guān)基因表達克隆構(gòu)建，華南地區(qū)特種疾病相關(guān)胚胎干細胞庫的建立。美國NIH（美國

2、國家衛(wèi)生研究院）：2019年7月-2019年1月，3000個斑馬魚克隆構(gòu)建。美國Novartis（美國諾華制藥）：2009年10月，美國諾華制藥簽約長期基因克隆供應(yīng)商。生命奧秘電子雜志2019年創(chuàng)刊，現(xiàn)已出版45期，擁有近30萬名讀者。一直關(guān)注最前沿資訊，關(guān)注內(nèi)容包括疾病療法、基因、蛋白、前沿技術(shù)等。誘導(dǎo)性多能干細胞腫瘤轉(zhuǎn)移RNAimicroRNA&癌癥染色質(zhì)與干細胞分化p53細胞信號通路GFP基因調(diào)控人類基因組測序40年抗癌歷程回顧蛋白動力學表觀基因組蛋白質(zhì)泛素化人類蛋白質(zhì)相互作用生物標志物新型蛋白標簽數(shù)量遺傳學疫苗安全與研發(fā)展望新型疫苗載體睡眠機制丙型肝炎抗腫瘤血管生成療法蛋白激酶與心血管

3、疾病HIV個性化用藥人類基因療法新一代DNA測序技術(shù)iPS技術(shù)光遺傳學技術(shù)海量生物學生物信息學在癌癥研究中的應(yīng)用核酸研究生物數(shù)據(jù)庫進展合成生物學轉(zhuǎn)化醫(yī)學組織工程與再生醫(yī)學Outline體細胞定向重編程的研究思路慢病毒的構(gòu)建原理慢病毒顆粒制備方法及應(yīng)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重編程因子的選擇靶細胞的選擇體細胞定向重編程重編程機制研究信號通路探討終末細胞的篩選和鑒定+檢測重編程效率終末細胞的功能指標檢測Profile Detection-qPCR ArrayProfile Detection-miRNA qPCR Array包含有65個miRNAs,可用于檢測干細胞的分化程度,同時也可篩選特異miRNAs用于體細胞重編程研究 fulengen/product/qpcr/mirna_array/miRNA qPCR Validated Primers人、小鼠、大鼠miRNA的Q-PCR引物都通過嚴格的實驗驗證fulengen/product/sea

6、rch/index.php?prt=22輸入miRNA name、Acc No或Sequence等即可獲得驗證結(jié)果Expression of reprogramming factors -ORF expression clone基因的ORF表達克隆有6種表達體系，100多種載體；該克隆可直接用于表達miRNA表達框已完全測序確證 基于慢病毒載體系統(tǒng)的表達克隆可轉(zhuǎn)染未分化細胞和難轉(zhuǎn)化的細胞，獲得與普通分化細胞類似的轉(zhuǎn)染效率 通過應(yīng)用eGFP可以監(jiān)測病毒和質(zhì)粒載體的轉(zhuǎn)染效率Expression of reprogramming factors -miRNA clonePre-made lenti-

7、vector expression clone已經(jīng)將約20,000條基因插入到慢病毒載體（Lv105）、哺乳動物載體（M02)等4套載體中，5個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理解細胞正常的分化機制，可以更準確地運用各種重編程因子進行細胞轉(zhuǎn)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慢病毒載體（Lentiviral Vector ）主要是以HIV-1（人類免疫缺陷I型病毒）為基礎(chǔ)發(fā)展起來的基因轉(zhuǎn)移載體 。HIV & replicationPackaging mix -four plasmids systemCMVgagpolRREpoly AEF1REVpoly

10、ACMVpoly AVSV-G包膜蛋白質(zhì)粒包裝質(zhì)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 將慢病毒顆粒稀釋 成不同濃度(0.02, 0.1, 1.0 and 5.0 ul)轉(zhuǎn)染細胞4872h后，在熒光顯微鏡下計數(shù)滴度=感染細胞數(shù)*病毒顆粒劑量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