关于慢病毒感染的相关知识总结

关于慢病毒感染的相关知识总结 慢病毒使用操作 一、慢病毒的储存与稀释: 1. 病毒的储存:收到病毒液后在很短时间内即使用慢病毒进行实验，可以将病毒暂时放置于4 ?保存;如需长期保存请放置于-80?(病毒置于冻存管，并使用封口膜封口) ? 病毒可以存放于-80? 6个月以上;但如果病毒储存时间超过6个月，建议在使用前需要重新滴定病毒滴度 ? 反复冻融会降低病毒滴度:每次冻融会降低病毒滴度10%;因此在病毒使用过程中应仅尽量避免反复冻融，为避免反复冻融,建议收到病毒后按照每次的使用量进行分装。 2. 病毒的稀释:如果需要稀释病毒，请将病毒取出置于冰浴融解后，使用培养目的细胞用PBS或无血清培养基(含血清或含双抗不影响病毒感染)。混匀分装后4?保存(请尽量在三天内用完) 分装后使用。 二、慢病毒用于体外(In Vitro)实验: 感染培养原代细胞和建系细胞。慢病毒对各种细胞和组织的亲嗜性不同，在使用慢病毒之前可以通过查阅相关文献，了解慢病毒对您的目的细胞的亲嗜性，感染复数(MOI 值)以及在体(In Vivo)注射所需要的病毒量。如果没有相关文献支持，可以通过感染预实验得到合适的感染复数(MOI 值)(使用24孔板检测病毒对目的细胞的亲嗜性)。 慢病毒感染目的细胞预实验 1. 慢病毒感染目的细胞预实验注意事项: ? 测定慢病毒对目的细胞的亲嗜性时，需要同时设置对慢病毒亲嗜性较高的细胞(HEK293T，Hela)作为平行实验的对照细胞。 ? 在进行慢病毒感染实验时，可以用完全培养基(培养目的细胞用)稀释;理论上，含有血清，双抗或者其他营养因子的完全培养基不影响慢病毒的感染效率。 ? 一般慢病毒单位为TU/ml，即每毫升中含有具有生物活性的病毒颗粒数。如:病毒 88滴度为>1X10 TU/ml 即每毫升病毒液中至少含有1X10个具有生物活性的慢病毒颗粒。 2. 以24孔培养板为例，进行目的细胞和HEK293T 细胞的感染预实验实验前按照不同的MOI设置不同的感染孔，并根据MOI和细胞数量计算所需要的病毒量，如有必要可以使用PBS溶液或者无血清培养基稀释病毒原液。 第一天，准备细胞:在24孔培养板接种若干孔，每个孔内接种3~5X104个目的细胞，铺板时细胞的融合率为50%左右，每孔培养基体积为100 μl;进行病毒感染时细胞的融合度约为70%左右。 第二天，准备病毒:取出4?保存的病毒，使用台式离心机离心20 秒(使病毒完全悬于离心管底部即可);如果是冻存在-80?的病毒需要先在冰上融化后使用。亦可以根据实验室的实际情况将按照MOI准确计算好的慢病毒稀释到培养基中，并尽可能保证所获得的含 有慢病毒的培养基的总体积为最小体积，以期获得最佳的感染效率。 第二天，感染目的细胞:病毒准备好之后，从培养箱中拿出细胞，首先察细胞生长状态，如细胞状态较好则开始实验。 a使用移液器吸取准确体积的病毒液加入准备好的培养基。 b吸去培养基的培养器皿中的培养基(如果细胞生长良好，密度适宜，则不用换液)。 c在目的细胞和对照细胞中分别加入计算好的病毒液。 d混匀后放于二氧化碳培养箱(37?、5%CO)孵育过夜。 2 注:感染前细胞的状态好坏对最终的感染效果高低影响很大，所以务必保证加病毒之前细胞处于良好的生长状态。亦可以将预先准备好的培养基和慢病毒的混合液直接加入培养器皿中。慢病毒对目的细胞的感染效率较低，通过提高MOI 值可以提高病毒的感染效率，但是当MOI高于20时，我们建议在培养基中加入ploybrene(8 μg/ml左右)来提高病毒的感染效率。 第三天，更换培养液:一般在24小时候后将含有慢病毒的培养液更换成正常培养液;在感染后观察细胞状态，如果慢病毒对细胞有明显毒性作用而影响细胞生长状态，可以最短在加病毒4小时候更换新鲜培养液后继续培养(建议在8-12小时更换为宜)。第一次换液后，如果慢病毒对细胞没有明显毒性作用，按照正常培养条件培养换液。 第六天，感染效率检测:在倒置荧光显微镜观察荧光，估计慢病毒感染目的细胞的效率;如何由于目的基因较大而造成选择的载体不能携带Marker Gene的，可以通过Real-timeRT-PCR检测目的基因的表达来拍评估感染效率。 注意: 有些慢病毒载体上带有GFP 绿色荧光蛋白，使用者可以在病毒感染96小时后用倒置荧光显微镜观察GFP 绿色荧光，以观察病毒对目的细胞的感染情况。如果慢病毒载体携带其他Marker Gene如RFP\BFP可以用荧光显微镜在对应的激发光波长下观察荧光表达的情况。 慢病毒表达时间较慢，荧光表达所需时间较长，建议感染后96小时后观测荧光表达。 感染后的细胞可以连续培养一周，通过观察荧光的表达时间和表达强度来确定慢病毒对目的细胞的感染情况。感染期间请根据细胞生长的情况对细胞进行及时换液，以保证细胞良好的生长状态。 三、慢病毒使用安全使用 慢病毒中的毒性基因已经被剔除并被外源性目的基因所取代，属于假型病毒因此没有毒性作用。但该病毒仍然具有可能的潜在的生物学危险，不建议使用编码已知或可能会致癌的基因的假型病毒。除非已经完全公认某个基因肯定没有致癌性，否则均不建议采用假型病毒进行生物学实验。使用时请参照如下所示进行实验: 1(病毒操作时最好使用生物安全柜。如果使用普通超净工作台操作病毒，请不要打开排风机。 2(病毒操作时请穿实验服，带口罩和手套。 samples if possible first enters the processing ... Sterilization containers: from plastic bags to sterilized gallon paint bucket, can be used to have a sharp edge products such as crab, shrimp, and so on. Sampling tools: sampling tool including: a teaspoon, spoon, needle-nosed pliers, angle fovceps tongs beakers and beaker, tool type is generally decided by the sample products. All sampling and date of sterilization of the container should be checked and sterilization time should be indicated on the label and packaging of equipment facilities, some facilities can be purchased at a local laboratory sterilization or disinfection equipment, laboratory and sterilization of instruments and facilities can be kept on the ground for at least two months, expired facilities must be sterilized again. Sterile gloves: sterile gloves must not be enabled in a sample, if a product is in the process of sample collection must be contacted to do it's best to let the factory production line workers (workers processed products), in the sample into a collection container, since the workers in the production process to reach the product, so we cannot think of their products and have the additional pollution. When the gloves must be a way to avoid contamination, wear, gloves must be fit to work needs. No bacteria cotton swab child: General for swab take instrument facilities and factory environment regional, using cotton swab child General has a right of program, open cotton swab child stripping off epidermal, then must carefully of put in tube head Shang, note don't contaminated cotton swab childliquid to dissolve in liquid. Often referred to as solvent of liquid components in the solution ...AVolume of water solution. 6. the titre (t) titer is the solution concentration is another method. It has two meanings, said on its per milliliter of solution of solute in grams or milligrams. Titration of sodium hydroxide solution NaoH = 0.0028g/mL=2.8m g/mL for t, second per ml of solution corresponds to the measured substance grams or milligrams. If titre of reagent T=3.5 card, 1mL card reagent is equivalent to 3.5 grams of water content, and when the determination of silver nitrate and sodium chloride, says there are two concentrations of silver nitrate: AgNO3 t t NaCl =1.84mg/mL, =1mg/mL, 1mg, indicating a 1mL solution containing silver nitrate, which represents the 1mL solution 1.84mg of sodium chloride, NaCl t =1.84 said, Known titre multiplied by the number of volume consumed in the titration of the standard solution, can be worked out of components to be measured, quite easy to calculate. Worth noting is that there are a lot of books or reagent also follows the concept of normality in the directory indicated by n, such as hydrochloric acid concentration is 0.1N indicates 1L hydrochloric acid solution containing 0.1 equivalent, also called volume parts per million. Was one of the original international concentration, is based on the equivalent law. Now with the new concept of "amount of substance, such as rules" instead of the equivalent law, so equivalent concentrations are no longer applied. On the relationship between n and m, the equivalent relation between concentration and Molarity, is not the same for different substances. Such as sulfuric acid: 1M h 2 SO 4 =2N h 2 SO 4, General writing m (1/2H 2 SO4) =0.1000mol/L, and potassium permanganate: 1M KMhO4 =5N KMnO 4, General writing m (1/5KMnO4) =0.1000mol/L. Third, the solution has not been made and saved (a) preparation of standard solution method 3(操作病毒时特别小心不要产生气雾或飞溅。如果操作时超净工作台有病毒污染，请立即用70%乙醇加1%的SDS 溶液擦拭干净。接触过病毒的枪头，离心管，培养板，培养液请于84 消毒液或1%SDS 中浸泡过夜后弃去。 4(用显微镜观察细胞感染情况时应遵从以下步骤:拧紧培养瓶或盖紧培养板。用70%乙醇清理培养瓶外壁后到显微镜处观察拍照。离开显微镜实验台之前，用70%乙醇清理显微镜实验台。 5(如需要离心，应使用密封性好的离心管，或者用封口膜封口后离心，而且尽量使用组织培养室内的离心机。 6(脱掉手套后，用肥皂和水清洗双手。 四、悬浮细胞感染方法概要 1. 根据细胞的量将细胞在1.5ml 管中离心收集然后用100-200ul 的无血清培养液稀释细胞沉淀，以细胞完全浸没在培养基中为准。 2. 按照MOI 换算病毒颗粒数量，吸取病毒液加入细胞中，将1.5ml 管放在37?度培养箱中孵育30 分钟。 3. 将管中混合溶液吸出加到培养皿中或孔里。 4. 加入足够量的新鲜培养液。 5. 12 小时后换液。 6. 96 小时后观察细胞阳性率。 五、相关专业术语: MOI:病毒感染复数传统的MOI 概念起源于噬菌体感染细菌的研究。其含义是感染时噬菌体与细菌的数量比值，也就是平均每个细菌感染噬菌体的数量。噬菌体的数量单位为pfu。一般认为MOI 是一个比值，没有单位，其实其隐含的单位是pfu number/cell。后来MOI 被普遍用于病毒感染细胞的研究中，含义是感染时病毒与细胞数量的比值，本手册中提到的MOI都是沿用这个概念。然而，由于病毒的数量单位有不同的表示方式，从而使MOI 产生了不同的含义。能产生细胞裂解效应的病毒例如单纯疱疹病毒等习惯上仍用pfu 表示病毒数量，因此其MOI 的含义与传统的概念相同。 六、细胞培养器皿的相关参数 Flask/Dish Surface (mm) Cell number Media Volume 96 well plate 50 1.5-5.0×10 100 μl 48 well plate 100 3.0×104-1.0×10 200 μl 24 well plate 200 8.0×104-2.0×10 500 μl 12 well plate 401 1.6-4.0×10 1.0 ml 6 well plate 962 3.0-8.0×10 2.0 ml 35mm 962 3.0-8.0×10 2.0 ml 60mm 2827 1.0-2.5×10 6.0 ml 100mm 7854 2.5-6.4×10 10.0 ml 慢病毒 重组慢病毒简介 在转染遗传物质到细胞基因组中的工作中，重组慢病毒载体是一个强大和有效的工具。慢病毒能作用于细胞周期的G/G期，同时具有嗜核性，所以可以感染分裂期细胞和非分裂期细胞，感染包括几乎所有01 的哺乳动物细胞、干细胞和原代培养的细胞。慢病毒载体具有携带基因片段容量大、转染效率高、可感染分裂细胞及非分裂细胞、目的基因可在宿主细胞中长时间稳定表达以及安全性好、免疫反应小等优点。所以获得了较广的应用范围。 慢病毒载体也是RNAi表达的主要手段，同化学合成和酶切形成的siRNA相比具有几个优点:其一，可以携带GFP或萤光素酶等报告基因共表达，便于跟踪、选择和富集转染的细胞;其二，可以根据需要表达不同类型的小RNA分子(siRNA、shRNA或miRNA);其三，具有较高的转染效率，使基因沉默维持较长时间。 目前Pubmed中收录有关慢病毒载体用作实验的文章超过7万多篇，仅Nature,，Science,，Cell上便超过600篇。已成为国际上最通用的转染系统，广泛用于各类实验室。 慢病毒的安全操作规范 深圳百恩维提供的慢病毒均采用第三代系统的慢病毒载体。水泡性口炎病毒来源的VSV-G基因代替HIV-1的env基因，这既提高了慢病毒的安全性，又使其能够感染的细胞种类大大增加。 本系统的慢病毒颗粒是“自身失活型(SIV)”，整合入靶细胞后，不会产生完整长度的RNA，仅仅具有携带目的基因的功能，感染目的细胞后不再感染其他细胞，也不会利用宿主细胞产生新的病毒颗粒。 尽管如此，该病毒仍然具有潜在的生物学危险性，因此建议不要使用编码已知或可能会致癌的基因的假性病毒;并强烈建议将本系统生产的慢病毒视为二级生物安全水平的生物，而且严格遵守BSL-2或 +BSL-2操作手册并进行相应的废弃物消毒。基本操作规范如下: 1、在实验室工作时，任何时候都必须穿着连体衣、隔离服或工作服;应戴上合适的手套和口罩。 2、病毒操作时最好使用生物安全柜。如果使用普通的超净工作台操作病毒，请不要打开风机。 3、所有的技术操作要按尽量减少气溶胶和微小液滴形成的方式来进行。如果出现病毒污染，请立即用70%的酒精加1%SDS溶液擦拭干净。 samples if possible first enters the processing ... Sterilization containers: from plastic bags to sterilized gallon paint bucket, can be used to have a sharp edge products such as crab, shrimp, and so on. Sampling tools: sampling tool including: a teaspoon, spoon, needle-nosed pliers, angle fovceps tongs beakers and beaker, tool type is generally decided by the sample products. All sampling and date of sterilization of the container should be checked and sterilization time should be indicated on the label and packaging of equipment facilities, some facilities can be purchased at a local laboratory sterilization or disinfection equipment, laboratory and sterilization of instruments and facilities can be kept on the ground for at least two months, expired facilities must be sterilized again. Sterile gloves: sterile gloves must not be enabled in a sample, if a product is in the process of sample collection must be contacted to do it's best to let the factory production line workers (workers processed products), in the sample into a collection container, since the workers in the production process to reach the product, so we cannot think of their products and have the additional pollution. When the gloves must be a way to avoid contamination, wear, gloves must be fit to work needs. No bacteria cotton swab child: General for swab take instrument facilities and factory environment regional, using cotton swab child General has a right of program, open cotton swab child stripping off epidermal, then must carefully of put in tube head Shang, note don't contaminated cotton swab childliquid to dissolve in liquid. Often referred to as solvent of liquid components in the solution ...AVolume of water solution. 6. the titre (t) titer is the solution concentration is another method. It has two meanings, said on its per milliliter of solution of solute in grams or milligrams. Titration of sodium hydroxide solution NaoH = 0.0028g/mL=2.8m g/mL for t, second per ml of solution corresponds to the measured substance grams or milligrams. If titre of reagent T=3.5 card, 1mL card reagent is equivalent to 3.5 grams of water content, and when the determination of silver nitrate and sodium chloride, says there are two concentrations of silver nitrate: AgNO3 t t NaCl =1.84mg/mL, =1mg/mL, 1mg, indicating a 1mL solution containing silver nitrate, which represents the 1mL solution 1.84mg of sodium chloride, NaCl t =1.84 said, Known titre multiplied by the number of volume consumed in the titration of the standard solution, can be worked out of components to be measured, quite easy to calculate. Worth noting is that there are a lot of books or reagent also follows the concept of normality in the directory indicated by n, such as hydrochloric acid concentration is 0.1N indicates 1L hydrochloric acid solution containing 0.1 equivalent, also called volume parts per million. Was one of the original international concentration, is based on the equivalent law. Now with the new concept of "amount of substance, such as rules" instead of the equivalent law, so equivalent concentrations are no longer applied. On the relationship between n and m, the equivalent relation between concentration and Molarity, is not the same for different substances. Such as sulfuric acid: 1M h 2 SO 4 =2N h 2 SO 4, General writing m (1/2H 2 SO4) =0.1000mol/L, and potassium permanganate: 1M KMhO4 =5N KMnO 4, General writing m (1/5KMnO4) =0.1000mol/L. Third, the solution has not been made and saved (a) preparation of standard solution method 4、如需离心，应使用密封性好的离心管，可用Parafilm膜封口后离心，而且最好使用组织或细胞培养室内的离心机。 5、用显微镜观察细胞感染情况时遵从以下步骤:拧紧培养瓶或盖紧培养板，并在使用70%乙醇清理所有受到污染的材料、标本和培养物在废弃或清洁再利用之前，必须清除污染。废弃的含病毒的培养基加入84消毒液(1:20左右)，浸泡一天后丢弃。接触过病毒的枪头，离心管，培养板等其他物品可用84消毒液稀释液处理，也可以用煮沸处理半小时或高压湿热灭菌(121?，30min)。 6、手套用完后，应先消毒再摘除，随后必须洗手。 详细操作规范请参阅卫生部发布的《微生物和生物医学实验室生物安全通用准则》(WS233-2002)、美国疾病控制中心出版的《微生物和生物医学实验生物安全(第五版)》和世界卫生组织出版《WHO生物安全手册》。操作慢病毒时请依照现有的使用指南。 包装细胞293T细胞 (下述流程来自深圳百恩维，如果使用不同公司系统请参考各公司提供的，本流程仅供参考) 293T细胞的冻存 1、随着传代的次数增加，293T细胞会出现生长状态下降，出现突变等，所以要在细胞购进时就进行备份。 2、在细胞对数生长期进行冻存，增加细胞复苏成活率。 3、倒去细胞上清液，加入D-Hank's液洗去残留的培养基。 4、加入0.25%的胰酶，消化10-20s后倒去。 5、镜下观察细胞变圆，细胞间间隙加大时，加入新鲜培养基吹打混匀。 6、细胞计数。 7、将细胞离心，1000rpm，2min。 68、根据计数结果加入细胞冻存液(70%完全培养基+20%FBS+10% DMSO)重悬细胞，密度为3×10个/ml。 10、第二天将细胞放入液氮灌，并记录。 293T细胞的传代 1、当细胞生长至汇合率达到80~90%需要对细胞进行传代操作，以扩大细胞数量，维持细胞良好的生长状态。 2、消化细胞，方法同上。 53、细胞离心结束后，加入完全培养基重悬。密度为3×10个/ml。 4、分到10cm培养皿中，10ml/皿。 293T细胞的复苏 1、当细胞传代次数过多(超过50代)，细胞状态变差时或细胞出现污染事故时，需要丢弃并对开始冻存的细胞进行复苏。 2、打开水浴锅，设置温度为37?。 3、查看细胞库记录，根据记录从液氮灌中取出冻存的细胞(需戴上棉手套，防止被冻伤)，迅速丢入水浴锅中并快速晃动，在1~2 min内使细胞溶液完全溶解。 4、将1ml细胞溶液加入9 ml完全培养基中并混匀后转入10cm培养皿。 5、放回37?、5%CO和95%相对湿度的培养箱中培养。 2 6、第二天观察细胞存活率。倒掉旧的培养基，加入10ml新鲜培养基。 慢病毒的包装、浓缩和滴度测定 1、所用病毒检测引物为WPRE特异引物，序列如下 5'-CCTTTCCGGGACTTTCGCTTT-3' (forward primer), 5'-GCAGAATCCAGGTGGCAACA-3' (reverse primer) and 5'-FAM-ACTCATCGCCGCCTGCCTTGCC-TAMRA-3' (probe) 2、TaqMan Universal PCR Master Mix (Applied Biosystems， cat. no. 4304437) 3、TaqMan DNA Template Reagent Kit (Applied Biosystems， cat. no. 401970) 4、TaqMan RNaseP control reagent (Applied Biosystems， cat. no. 4316844) 用于包装的293T细胞(ATCC No. CRL-11268)必需选择处于生长旺盛期，细胞状态较佳，存活率90%以上，细胞边缘清晰，传代次数较低。任何时候细胞汇合率都不准达到100%。 慢病毒的包装 1、预先准备3个T150瓶的293T细胞，培养基为DMEM + 10% FBS，1% Glutamax ，1% 青霉素-链霉素。 62、将细胞分到12个T150瓶中，每瓶的细胞密度是8×10个。 3、第二天，镜下检查细胞。细胞融合度应大致为30-40%，分布均匀。 4、转染前1小时，取出细胞板，去除原有细胞培养基，加入20ml的Opti-MEM培养基，将细胞送回培养箱。 5、取两支无菌的50ml离心管，其中一支中加入252 μg pNL-EGFP/CMV/WPREDU3 载体质粒，168 μg pCD/NL-BH*DDD 包装质粒和84 μg pLTR-G质粒，用Opti-MEM培养基补齐到18ml。另一支中加入500 μl Trans-EZ溶液和17.5 ml Opti-MEM培养基，用电动移液器轻轻混匀。将Trans-EZ稀释液滴加到质粒管中，边加边轻轻晃匀。 关键步骤:推荐使用Qiagen质粒大抽试剂盒或相当的试剂盒所制备的质粒。 6、室温孵育20分钟，使DNA和Trans-EZ充分结合形成转染复合体。 7、取1支5ml的移液管，将得到的DNA-Trans-EZ复合体均匀滴加入到细胞培养板中，每板3ml。来回晃动培养板，混匀后放回到5%二氧化碳培养箱。每一皿细胞培养盘不要超过6盘。 8、6小时后，移去细胞上清，更换为17ml的DMEM完全培养基。 9、转染后一天观察细胞，所有的细胞应当都是健康的并且密度接近60-80%。如果所转染质粒带有GFP荧光，那么这时候可以看到大于95%的细胞都是带有荧光的。 10、将细胞送回培养箱继续培养2天(36-48小时)。在这个过程中，细胞会逐渐融合形成多核体，大多数的细胞依然贴壁。 samples if possible first enters the processing ... Sterilization containers: from plastic bags to sterilized gallon paint bucket, can be used to have a sharp edge products such as crab, shrimp, and so on. Sampling tools: sampling tool including: a teaspoon, spoon, needle-nosed pliers, angle fovceps tongs beakers and beaker, tool type is generally decided by the sample products. All sampling and date of sterilization of the container should be checked and sterilization time should be indicated on the label and packaging of equipment facilities, some facilities can be purchased at a local laboratory sterilization or disinfection equipment, laboratory and sterilization of instruments and facilities can be kept on the ground for at least two months, expired facilities must be sterilized again. Sterile gloves: sterile gloves must not be enabled in a sample, if a product is in the process of sample collection must be contacted to do it's best to let the factory production line workers (workers processed products), in the sample into a collection container, since the workers in the production process to reach the product, so we cannot think of their products and have the additional pollution. When the gloves must be a way to avoid contamination, wear, gloves must be fit to work needs. No bacteria cotton swab child: General for swab take instrument facilities and factory environment regional, using cotton swab child General has a right of program, open cotton swab child stripping off epidermal, then must carefully of put in tube head Shang, note don't contaminated cotton swab childliquid to dissolve in liquid. Often referred to as solvent of liquid components in the solution ...AVolume of water solution. 6. the titre (t) titer is the solution concentration is another method. It has two meanings, said on its per milliliter of solution of solute in grams or milligrams. Titration of sodium hydroxide solution NaoH = 0.0028g/mL=2.8m g/mL for t, second per ml of solution corresponds to the measured substance grams or milligrams. If titre of reagent T=3.5 card, 1mL card reagent is equivalent to 3.5 grams of water content, and when the determination of silver nitrate and sodium chloride, says there are two concentrations of silver nitrate: AgNO3 t t NaCl =1.84mg/mL, =1mg/mL, 1mg, indicating a 1mL solution containing silver nitrate, which represents the 1mL solution 1.84mg of sodium chloride, NaCl t =1.84 said, Known titre multiplied by the number of volume consumed in the titration of the standard solution, can be worked out of components to be measured, quite easy to calculate. Worth noting is that there are a lot of books or reagent also follows the concept of normality in the directory indicated by n, such as hydrochloric acid concentration is 0.1N indicates 1L hydrochloric acid solution containing 0.1 equivalent, also called volume parts per million. Was one of the original international concentration, is based on the equivalent law. Now with the new concept of "amount of substance, such as rules" instead of the equivalent law, so equivalent concentrations are no longer applied. On the relationship between n and m, the equivalent relation between concentration and Molarity, is not the same for different substances. Such as sulfuric acid: 1M h 2 SO 4 =2N h 2 SO 4, General writing m (1/2H 2 SO4) =0.1000mol/L, and potassium permanganate: 1M KMhO4 =5N KMnO 4, General writing m (1/5KMnO4) =0.1000mol/L. Third, the solution has not been made and saved (a) preparation of standard solution method 11、收集所有的上清，分装到50ml离心管中。 12、4?，500g离心10分钟，除去脱落的细胞和大的细胞碎片。 13、总的上清约为204 ml，用250-ml 0.45 μm PVDF过滤装置过滤。如果发现滤膜被堵住，表现为过滤速度变慢，更换新的滤器。 慢病毒的浓缩与纯化 方法一:超速离心沉淀法 1、取6个Ultra-clear SW28离心管，用70%乙醇消毒后，放在超净工作台中打开紫外灯继续消毒30分钟。 2、每个Ultra-clear SW28离心管中加入约32ml的预先处理的病毒上清液。 3、取一支10ml的移液管，吸取12 ml 20%的蔗糖溶液。将移液管一直插入到离心管的底部，缓慢将蔗糖溶液打出4 ml。同样地，将剩下8 ml的蔗糖溶液分别加入到另两个离心管中。另取一支干净的移液管，对剩下3管进行同样处理。 4、用PBS调整各管的重量，使对应的离心管之间的重量相差不超过0.1g。 5、按次序将所有6个离心管放入Beckman SW28 超速离心转头中。 6、小心将管子从转头中取出。倒掉上清，将离心管倒扣在纸巾上放置10分钟使剩余的上清流干。吸掉剩余的液滴。在管底应当有可见的沉淀。 7、每管中加入100ml 不含钙和镁的PBS洗下沉淀。 8、将SW28超速离心管插入到50ml锥底离心管中，盖上盖子。 9、在4?溶解2小时，每隔20分钟轻轻震荡。 10、4?，500g离心1分钟，使溶液集中于管底。 11、用200μl 移液器轻柔吹打使沉淀重悬。避免产生泡沫。将所有管中的液体集中到一个SW28离心管中。 12、集中后的病毒悬液分装成50μl 每份，保存在成品管中。用碎干冰速冻后储存在-80 ?。 方法二 PEG-8000浓缩法 5X PEG8000+NaCl配制称取NaCl 8.766 g; PEG8000 50g溶解在200ml Milli-Q纯水中;121摄氏度 30min 湿热灭绝 30min;保存在4?。 1、使用0.45μm滤头过滤慢病毒上清液; 2、每20,30min混合一次，共进行3-5次; 3、4度放置过夜; 4、4度，4000 g，离心 20min; 5、吸弃上清，静置管子1,2分钟，吸走残余液体; 6、加入适量的慢病毒溶解液溶解慢病毒沉淀; 7、集中后的病毒悬液分装成50 μl每份，保存在成品管中。用碎干冰速冻后储存在-80 ? 病毒滴度的测定 稀释计数法 滴度单位:TU/ml，指每毫升中含有的具有生物活性的病毒颗粒数。”TU”为”transducing units”的缩写，中文为“转导单位”，表示可以感染并进入到靶细胞中的病毒基因组数。 第一天细胞准备 5将生长状态良好的293T细胞消化计数后稀释至1×10/ml，加入96孔板，100μl/孔，为每个病毒准备10个孔。放入37?，5%CO培养箱中培养。 2 第二天加病毒 在EP管中做10倍梯度稀释，连续10个稀释度。稀释方法如下:每种病毒准备10个1.5ml EP管，每管加入90μl培养液，往第一个管中加入10μl病毒原液，混匀后，吸取10μl加入第二个管混匀。依此类 -8推，做十个稀释度(10~10)。 吸取96孔板中原有的培养基，加入含稀释好的病毒液。并做好标记。 第三天追加培养液 在每个孔再加入100μl完全培养液，利于细胞的生长。 第五天观察结果并计算滴度 在荧光显微镜下观察结果，并数出最后两个有荧光的荧光细胞克隆数。假设为X和Y，则滴度(TU/ml)=(X+Y*10)*1000/2/X孔的病毒液的含量(μl)。 定量PCR法 4病毒感染1天前，取6孔板接种HOS细胞，每孔细胞为5×10个。 接种细胞24小时后，取两个孔的细胞用血球计数板计数，确定感染时细胞的实际数目，记为N。 弃去其他培养板中的培养基，更换为含有5μg/ml polybrene的新鲜培养基。将浓缩病毒用培养基稀释200倍，也就是取1μl病毒加入到199μl的培养基中。在3个培养孔中分别加入0.5μl，5μl和50μl的稀释病毒。 感染开始后20小时，除去培养上清，换为500μl含DNaseI (Takara Mirus Bio，终浓度为10 U/ml)的新鲜培养基。在37?消化15分钟，这一步是要除去残余的质粒DNA。然后换为2ml正常的培养基，继续培养48小时。 用0.5ml 0.25%胰酶-EDTA溶液消化细胞，在37?放置1分钟。用培养基吹洗下，离心收集细胞。按照DNeasy试剂盒的说明抽提基因组DNA。每个样品管中加入200μl洗脱液洗下DNA。用DNA定量试剂盒定量(Bio-Rad)。基因组DNA可以稳定保存在-20?至少2个月。 准备PCR所需的试剂和样品。为病毒序列检测引物配总管?: 25 μl × 2× TaqMan Master Mix n Forward primer (100 0.1μl × pmol ml-1) n Reverse primer (100 0.1μl × pmol ml-1) n 0.1μl × Probe (100 pmol ml-1) n 19.7μl HO 2× n samples if possible first enters the processing ... Sterilization containers: from plastic bags to sterilized gallon paint bucket, can be used to have a sharp edge products such as crab, shrimp, and so on. Sampling tools: sampling tool including: a teaspoon, spoon, needle-nosed pliers, angle fovceps tongs beakers and beaker, tool type is generally decided by the sample products. All sampling and date of sterilization of the container should be checked and sterilization time should be indicated on the label and packaging of equipment facilities, some facilities can be purchased at a local laboratory sterilization or disinfection equipment, laboratory and sterilization of instruments and facilities can be kept on the ground for at least two months, expired facilities must be sterilized again. Sterile gloves: sterile gloves must not be enabled in a sample, if a product is in the process of sample collection must be contacted to do it's best to let the factory production line workers (workers processed products), in the sample into a collection container, since the workers in the production process to reach the product, so we cannot think of their products and have the additional pollution. When the gloves must be a way to avoid contamination, wear, gloves must be fit to work needs. No bacteria cotton swab child: General for swab take instrument facilities and factory environment regional, using cotton swab child General has a right of program, open cotton swab child stripping off epidermal, then must carefully of put in tube head Shang, note don't contaminated cotton swab childliquid to dissolve in liquid. Often referred to as solvent of liquid components in the solution ...AVolume of water solution. 6. the titre (t) titer is the solution concentration is another method. It has two meanings, said on its per milliliter of solution of solute in grams or milligrams. Titration of sodium hydroxide solution NaoH = 0.0028g/mL=2.8m g/mL for t, second per ml of solution corresponds to the measured substance grams or milligrams. If titre of reagent T=3.5 card, 1mL card reagent is equivalent to 3.5 grams of water content, and when the determination of silver nitrate and sodium chloride, says there are two concentrations of silver nitrate: AgNO3 t t NaCl =1.84mg/mL, =1mg/mL, 1mg, indicating a 1mL solution containing silver nitrate, which represents the 1mL solution 1.84mg of sodium chloride, NaCl t =1.84 said, Known titre multiplied by the number of volume consumed in the titration of the standard solution, can be worked out of components to be measured, quite easy to calculate. Worth noting is that there are a lot of books or reagent also follows the concept of normality in the directory indicated by n, such as hydrochloric acid concentration is 0.1N indicates 1L hydrochloric acid solution containing 0.1 equivalent, also called volume parts per million. Was one of the original international concentration, is based on the equivalent law. Now with the new concept of "amount of substance, such as rules" instead of the equivalent law, so equivalent concentrations are no longer applied. On the relationship between n and m, the equivalent relation between concentration and Molarity, is not the same for different substances. Such as sulfuric acid: 1M h 2 SO 4 =2N h 2 SO 4, General writing m (1/2H 2 SO4) =0.1000mol/L, and potassium permanganate: 1M KMhO4 =5N KMnO 4, General writing m (1/5KMnO4) =0.1000mol/L. Third, the solution has not been made and saved (a) preparation of standard solution method n = number of reactions. 例如:总反应数为40，将1ml 2× TaqMan Universal PCR Master Mix，4μl forward primer，4μl reverse primer，4μl probe 和788μl HO混和。震荡后放在冰上。 2 为人基因组序列检测引物配总管?: 2× TaqMan Master 25 μl Mix × n 10×RNaseP 2.5 μl primer/probe mix × n 17.5μHO 2l × n n = number of reactions. 例如:总反应数为40，将1ml 2× TaqMan Universal PCR Master Mix，100μl 10×RNaseP primer/probe mix和700μl HO混和。震荡后放在冰上。 2 在预冷的96孔PCR板上完成PCR体系建立。从总管?中各取45μl加入到A-D各行的孔中，从总管?中各取45μl加入到E-G各行的孔中。 分别取5μl质粒品和待测样品基因组DNA加入到A-D行中，每个样品重复1次。另留1个孔加入5μl的水做为无模板对照(no-template control)。 分别取5μl基因组标准品和待测样品基因组DNA加入到E-G行中，每个样品重复1次。另留1个孔加入5μl的水做为无模板对照(no-template control)。 所使用定量PCR仪为ABI PRISM 7000定量系统。循环条件设定为: 50? 2分钟，95? 10分钟，然后是95? 15秒，60? 1分钟的40个循环。 数据分析:测得的DNA样品中整合的慢病毒载体拷贝数用基因组数加以标定，得到每基因组整合的病毒拷贝数。 -1滴度(integration units per ml，IU ml)的计算如下: IU ml-1 = (C ×N× D×1000)/V 其中: C = 平均每基因组整合的病毒拷贝数 5 N = 感染时细胞的数目(约为1×10) D = 病毒载体的稀释倍数 V = 加入的稀释病毒的体积数 慢病毒的储存与稀释 慢病毒的储存 1、病毒的运输采用干冰保温，收到病毒液后若几天内用于实验，可于4?保存(于一周内用完)。 2、若病毒量较大，需长期保存。则根据每次实验用量分装后放于-80?冰箱。一般病毒可以放于-80?约12个月以上，但若超过6个月后使用，请重新检测病毒滴度。 3、避免反复冻融，否则会降低病毒滴度。每次冻融会降低病毒滴度10%。 慢病毒的稀释 需要稀释病毒时，将病毒取出后置于冰上融解，用细胞培养用D-Hank's、PBS或培养基稀释到所需浓度后混匀分装后4?保存，并尽快用于实验(于一周内用完)。 慢病毒在细胞水平的使用 什么是MOI, “MOI”为”multiplicity of infection”的缩写，中文为“感染复数”或“复感染指数”，含义为感染时病毒和细胞数量的比值，即平均每个细胞感染的病毒活性单位数(TU number /cell)。在实验中将某种细胞感染达到80%时的MOI定义为这种细胞的MOI。 MOI与整合事件以及目的基因的表达相关。一定范围内，表达水平和MOI呈正相关。MOI取决于多种因素，如细胞状态，目的基因的大小与性质，细胞的感染效率等。所以，实验前需查阅相关文献，确定慢病毒对目的细胞的亲嗜性、MOI以及在体(in vivo)注射所需病毒量。若无文献支持，可以通过预实验得到合适的MOI。实际上，即使有文献支持，但由于所用细胞代数、细胞状态以及目的基因的差异，实际的MOI和文献报道也会不同，所以要安排预实验以确定所需MOI以及病毒对细胞生长的影响。 目的细胞感染预实验及实验体系的放大 实验目的:确定细胞感染所需的MOI，以及是否需要添加感染增强剂Polybrene。 实验材料:96孔板，1.5ml EP管，长势良好的目的细胞一瓶，已知滴度的慢病毒溶液，Polybrene(10mg/ml)，目的细胞培养基等。 第一天:细胞准备 将长势良好的目的细胞接种到96板，消化好细胞(悬浮细胞不需要消化，直接离心收集细胞后加新 4~54鲜培养基重悬即可)后把浓度调为3×10×10个/ml，按90μl/孔加入。接种细胞数量因细胞的生长速度而略有不同，一般是保证第二天进行病毒感染时细胞汇合率介于30~50%之间。 第二天:病毒感染 感染实验分两组，一组直接添加病毒液，一组同时添加Polybrene。病毒稀释方法同病毒滴度测定时 4方法，10倍倍比稀释，共三个稀释度，MOI值依次为100，10和1(细胞经过一天的生长数量约为1×10 654个/孔，对应的病毒数量为1×10TU、1×10TU和1×10TU)。每个MOI值加两个孔，取出一组加入感染增强剂，比例为1:2000，终浓度为5μg/ml。 第二天:换液 感染实验同一天，8-12小时后，弃去上清液，更换为新鲜培养基，100μl/孔。 第五天:观察荧光表达情况 在倒置荧光显微镜下观察荧光，估计慢病毒感染目的细胞的效率。对于生长缓慢的细胞，可以适当推迟观察的时间，中途可以传代和换液，以保持细胞良好的生长状态。通过细胞感染的效果，确认目的细胞的感染MOI以及是否需要添加Polybrene。 对于因目的基因较大，载体无法携带标志基因的，可以通过定量PCR来检测目的基因的表达来评估感染效率。 实验体系的放大 正式实验时，由于细胞数量较大，所需的病毒用量也需相应增大。放大的原则是保持细胞的密度不变，将培养基体积，病毒量按实际细胞数与预实验细胞数的比例放大。即使这样，正式实验时由于体系的改变，加上预实验的MOI值设置跨度较大，可进行对MOI的再次优化。MOI的设置值为预实验最佳值0.5倍，1倍和1.5倍或自己设置合理的数值。 samples if possible first enters the processing ... Sterilization containers: from plastic bags to sterilized gallon paint bucket, can be used to have a sharp edge products such as crab, shrimp, and so on. Sampling tools: sampling tool including: a teaspoon, spoon, needle-nosed pliers, angle fovceps tongs beakers and beaker, tool type is generally decided by the sample products. All sampling and date of sterilization of the container should be checked and sterilization time should be indicated on the label and packaging of equipment facilities, some facilities can be purchased at a local laboratory sterilization or disinfection equipment, laboratory and sterilization of instruments and facilities can be kept on the ground for at least two months, expired facilities must be sterilized again. Sterile gloves: sterile gloves must not be enabled in a sample, if a product is in the process of sample collection must be contacted to do it's best to let the factory production line workers (workers processed products), in the sample into a collection container, since the workers in the production process to reach the product, so we cannot think of their products and have the additional pollution. When the gloves must be a way to avoid contamination, wear, gloves must be fit to work needs. No bacteria cotton swab child: General for swab take instrument facilities and factory environment regional, using cotton swab child General has a right of program, open cotton swab child stripping off epidermal, then must carefully of put in tube head Shang, note don't contaminated cotton swab childliquid to dissolve in liquid. Often referred to as solvent of liquid components in the solution ...AVolume of water solution. 6. the titre (t) titer is the solution concentration is another method. It has two meanings, said on its per milliliter of solution of solute in grams or milligrams. Titration of sodium hydroxide solution NaoH = 0.0028g/mL=2.8m g/mL for t, second per ml of solution corresponds to the measured substance grams or milligrams. If titre of reagent T=3.5 card, 1mL card reagent is equivalent to 3.5 grams of water content, and when the determination of silver nitrate and sodium chloride, says there are two concentrations of silver nitrate: AgNO3 t t NaCl =1.84mg/mL, =1mg/mL, 1mg, indicating a 1mL solution containing silver nitrate, which represents the 1mL solution 1.84mg of sodium chloride, NaCl t =1.84 said, Known titre multiplied by the number of volume consumed in the titration of the standard solution, can be worked out of components to be measured, quite easy to calculate. Worth noting is that there are a lot of books or reagent also follows the concept of normality in the directory indicated by n, such as hydrochloric acid concentration is 0.1N indicates 1L hydrochloric acid solution containing 0.1 equivalent, also called volume parts per million. Was one of the original international concentration, is based on the equivalent law. Now with the new concept of "amount of substance, such as rules" instead of the equivalent law, so equivalent concentrations are no longer applied. On the relationship between n and m, the equivalent relation between concentration and Molarity, is not the same for different substances. Such as sulfuric acid: 1M h 2 SO 4 =2N h 2 SO 4, General writing m (1/2H 2 SO4) =0.1000mol/L, and potassium permanganate: 1M KMhO4 =5N KMnO 4, General writing m (1/5KMnO4) =0.1000mol/L. Third, the solution has not been made and saved (a) preparation of standard solution method 表1.病毒感染细胞所用培养基体积和病毒量参考值 单孔感染正常培MOI=1 MOI=10 MOI=10底面积时体积 养体积(μl) 病毒量(TU) 病毒量(TU) 0病毒量(TU (cm2) (μl) 96444 0.3 100 100 1×10 1×10 1×10 孔板 484560.6 200 200 2×10 2×10 2×10 孔板 24456 1.9 500 500 6×10 6×10 6×10 孔板 12567 3.8 1000 500 2×10 6×10 6×10 孔板 65679.5 2000 1000 2×10 2×10 2×10 孔板 T56725 5000 2500 5×10 5×10 5×10 25瓶 *表中可培养板底参数数值为CORNING公司产品参数。 *对于孔面积较小的培养板，由于溶液张力的关系，培养基体积太少会导致病毒的分布不均与，所以不做减半处理。 慢病毒在整体动物水平的使用 到目前为止，慢病毒已经广泛应用于大鼠，小鼠，兔，猪和鸡等动物的局部注射和基因治疗等领域，所采用的方法包括皮下注射，尾静脉注射和核团注射等，也可以先对原代细胞或者细胞系进行基因操作之后再回输体内进行治疗实验，一般流程如下图所示: 由于慢病毒在整体动物实验的复杂性和多样性，请直接将您的问题和建议与我们的动物工程师讨论，深圳百恩维动物中心的慢病毒技术工程师也愿意与您分享他们在这一领域的经验。