Lenti-CMV-EGFP-p65-EF1α-Puro

Lenti-CMV-EGFP-p65-EF1α-Puro，即Lentivirus expressing EGFP-p65 fusion protein and puromycin，是阿拉丁自行研发的一种可以在大多数哺乳动物细胞(包括原代细胞和干细胞)中通过CMV启动子表达EGFP-p65融合蛋白的重组慢病毒。本产品可用于检测NF-κB信号通路的激活和抑制。本产品主要适合用于通过荧光显微镜等观察NF-κB的核转运激活。本产品带有嘌呤霉素(puromycin)抗性，感染细胞后可以筛选稳定株。NF-κB (Nuclear factor kappa-light-chain-enhancer of activated B cells)，中文名为激活的B细胞核因子kappa-轻链增强子，是细胞内重要的核转录因子，参与细胞对外界刺激的响应、炎症反应、免疫应答等过程，几乎存在于所有类型的动物细胞中。NF-κΒ作为转录因子蛋白家族，包括5个亚单位：p50 (NF-κB1)、p52 (NF-κB2)、p65 (REL-associated protein, Rel-A)、Rel-B和c-Rel，它们的N端都具有高度保守的RHD (REL homology domain)；5个亚单位根据C端结构域的不同被分为两类，第一类为p50、p52，其C端具有ankyrin重复结构域具有转录抑制活性(Transrepression activity)和招募IKK (Inhibitory κB kinase)的致死结构域(Dead domain)；第二类为p65、Rel-B和c-Rel，其C端具有转录所必需的转录激活结构域(Transactivation domain, TAD)1]。NF-κB在静息状态或失活状态(Resting state or inactive state)下，其高度保守的RHD中的核定位信号(Nuclear localization sequence, NLS)与抑制因子IκB (Inhibitor of NF-κB)结合形成非活性复合物，NF-κB以失活状态存在于细胞质中。虽然IκB蛋白掩盖了p65的NLS信号，但没有掩盖p50的NLS信号，因此静息状态下NF-κB可以持续在细胞核和细胞质之间穿梭(Shuttle)维持很低水平的活性2]。NF-κB的激活存在两条途径，即经典途径和非经典途径。经典途径：在受到促炎信号(如炎症细胞因子TNF-α、IL-1、病原体和病毒双链RNA等)的刺激后，使细胞中的IKK复合物被招募并激活，IKK复合物由IKKα和/或IKKβ催化亚基以及NEMO (NF-κB essential modulator，亦称IKKγ)组成，IKK复合物使IκB被磷酸化，磷酸化的IκB随后发生泛素化被蛋白酶体降解，使得NF-κB的核定位信号暴露从而进入细胞核激活靶基因；非经典途径：该途径由NF-κB抑制激酶NIK (NF-κB inhibitory kinase)介导，NIK磷酸化并激活IKK的亚基IKKα，磷酸化的IKKα再使p100磷酸化，导致加工和释放有活性的p52和RelB异源二聚体转移至细胞核并激活靶标基因，这一过程发生在淋巴器官发育期间，淋巴器官负责产生B淋巴细胞和T淋巴细胞2]。Lenti-CMV-EGFP-p65-EF1α-Puro感染HEK293T细胞的效果如图1所示。图1.阿拉丁Lenti-CMV-EGFP-p65-EF1α-Puro感染HEK293T细胞的效果图。96孔板每孔10,000个细胞，培养过夜后用相应的病毒TU数进行感染，病毒感染72小时后荧光显微镜实拍相同大小视野效果图。静息状态下，细胞质显示明亮的绿色荧光。实际检测效果会因检测仪器、实验条件的不同而存在差异，本图仅供参考。慢病毒感染细胞后会将目的基因随机整合到基因组DNA上，因而可以长期稳定地表达目的蛋白，并且几乎可以感染所有哺乳动物细胞，在很多不分裂的细胞中也可以长期稳定表达，广泛应用于细胞和动物实验。本产品表达嘌呤霉素抗性基因，因此本产品感染培养的细胞后可以使用嘌呤霉素筛选稳定表达的细胞株以用于后续研究。阿拉丁的Lenti-CMV-EGFP-p65-EF1α-Puro是复制缺陷型慢病毒。其3' LTR的增强子功能发生缺失，形成了自失活(self-inactivating) 3' LTR，并且5' LRT中的U3区域替换成CMV启动子，在感染普通的细胞后不能进行复制和扩增，从而有效降低了本产品在活体生物中的风险。阿拉丁推荐的慢病毒感染不同种类体外培养细胞的MOI值参见下表。实验室常用的逆转录病毒(Retrovirus)、慢病毒(Lentivirus)、腺相关病毒和腺病毒(Adenovirus)的主要特征之间的比较和差别参见下表。具体的特定病毒的一些特征和下表相比可能会有一定差异。本产品的滴度为10^9TU/ml，适合细胞实验或活体动物实验。TU, transduction unit，即转导单位。TU通过本产品梯度稀释后感染HEK293T细胞后测定获得，能让一个HEK293细胞呈现绿色荧光的活力单位定义为1TU。MOI (Multiplicity of Infection)是病毒感染细胞时，病毒数量与细胞数量的比值。使用10^9TU/ml的本产品，如果按照5 MOI感染6孔板的细胞，每孔50万细胞计算，100μl共可以感染40个孔；如果按照5 MOI感染24孔板的细胞，每孔10万细胞计算，100μl共可以感染200个孔。如果MOI值提高，那么相应可以感染的孔数会减少；如果MOI值下调，那么相应可以感染的孔数会增加。 注意事项 ： 反复冻融会降低病毒滴度，如有必要请在收到本产品后分装保存。分装时必须在冰浴上进行。病毒融解后，如果在一周内使用，可以放置于4℃，但须注意4℃存放时间越长，滴度下降越明显。如果-80℃保存时间超过一年，可能会导致滴度下降，此时建议重新测定病毒滴度。本产品使用前请仔细阅读附录1《慢病毒使用安全规范》。本产品生物安全等级为Biosafety Level 2 (BSL-2)，在按照常规的微生物实验操作要求进行操作的基础上，还需要注意限制接触、生物危害提示、显著的警示标识、并制定相应的安全规范。病毒操作中应注意有效防护，绝对禁止在生物安全柜内有任何皮肤直接暴露的情况。实验完成后，请及时清洗双手。严禁直接接触病毒，如意外接触，请及时用清水冲洗，并适当用70%乙醇对皮肤进行消毒。任何接触过病毒的材料、试剂、样品，应经消毒处理，可以采用1%的SDS溶液、或84消毒液(1:20)浸泡30分钟以上，或121℃高压灭菌30分钟。本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。为了您的安全和健康，请穿实验服并戴一次性手套操作。 使用说明 ： 1.感染条件的确定：MOI (Multiplicity of Infection)定义：病毒感染细胞时，病毒数量与细胞数量的比值。TU (Transduction Units)定义：具有生物活性的病毒颗粒数量。梯度稀释病毒后感染细胞，根据出现荧光的细胞数量或抽提细胞基因组DNA进行qPCR测定来确定具有生物活性的病毒颗粒数量。不同种类的细胞感染所需的MOI值是不同的，如果是初次使用慢病毒感染需要通过实验确定最佳的感染条件。理论上MOI值越高，慢病毒感染可能性越大，感染效率越高，对细胞的毒性也越大。所以预实验的目的是在保证细胞成活率的情况下，确定能够使感染效率达到适宜观察水平的MOI值。a.细胞培养(以24孔板HEK293T细胞为例，其它培养板或培养皿参考24孔板进行操作)：感染前一天，在24孔板中以约5×104/孔接种HEK293T细胞，每孔加入0.5ml完全培养液，使第二天病毒感染时的细胞密度达到约70-80% (细胞数约1×105)。注：具体接种数量需根据细胞种类、细胞大小和细胞生长速度等因素而确定。b.设置MOI值分别为1、2、5、10、20，计算所需病毒量，计算公式如下：TU = 感染时的细胞数* × MOI病毒母液(μl) = TU / 滴度(TU/ml) × 1000*一般第二天细胞数按接种细胞数增长1倍来计算，增殖较慢或不增殖的细胞可适当调整。例如：需要向1×105个HEK293T细胞加入20 MOI的病毒，即所需TU = (1×105 cells) × (20 MOI) = 2×106 TU。若病毒母液滴度为1×109 TU/ml，则细胞培养液中应加入病毒母液 = 2×106 TU / (1×109 TU/ml) × 1000 = 2μl，即2μl病毒母液。注：其它细胞株的预实验MOI设置可以参考本说明书中产品简介部分“阿拉丁推荐的慢病毒感染不同种类体外培养细胞的MOI值”表格或相关文献资料。c.按照下图设置MOI预实验组，建议设置复孔以确保实验准确性。注：对于适合使用聚凝胺(polybrene) 处理的细胞，弃旧培养液，每孔加入250μl含有6-8μg/ml polybrene的新鲜培养液；对于不适合使用polybrene处理的细胞，弃旧培养液，每孔加入250μl的新鲜培养液。使用polybrene可以使感染效率提高约2-10倍，并且病毒测定其活力的时候是使用polybrene的。须特别注意此时宜加入尽量少的新鲜培养液，以提升后续病毒的感染效率。 图2.细胞感染MOI值预实验设计分组。MOI依次设置为：1、2、5、10、20。并同时设置加含6-8μg/ml polybrene的培养液实验组，不含polybrene的培养液实验组及Blank组(空白对照组)。Blank组可作为参照，以检验细胞生长状态。本图中实验设置仅供参考，用户应根据实际情况自行适当调整。d.取出本产品置于冰上解冻，混匀后，将根据特定MOI值计算好的病毒母液量分别加入细胞培养板中，轻轻混匀后继续培养。注：如果病毒滴度太高，需加的病毒母液量较少或多孔板如96孔板和48孔板等培养液体积比较小的情况，可以先将病毒母液用培养液进行适当稀释后再加入。e.感染后约4小时，每孔补加250μl新鲜培养液。f.感染后约24小时，进行换液。弃含病毒的培养液，每孔加入新鲜的完全培养液，继续培养。注：换液的具体时间需视细胞状态而定，如果慢病毒对细胞有明显毒性作用，影响细胞生长状态，最短可于加病毒4小时后更换新鲜培养液。g. 继续培养约48-96小时后观察细胞生长状况及荧光蛋白表达情况，以不显著影响细胞生长、荧光较强且感染效率便于荧光观察的MOI值和感染后时间为最佳条件。h.如果有必要，可以根据特定细胞加入适当浓度的嘌呤霉素(Puromycin) ，以筛选被感染的细胞，后续可以通过稀释法等获取单克隆细胞株。嘌呤霉素的浓度可以通过梯度稀释的预实验来确定，以刚好能完全杀死目的细胞的浓度为宜。注1：细胞并非感染效率越高越好，通常约20-70%的感染效率已经足以进行后续检测，感染效率过高反而容易产生细胞毒性而干扰检测。注2：通常在慢病毒感染细胞后72小时即可观察到荧光蛋白表达的出现，在96小时左右有较强的表达效果。注3：对于感染慢病毒后出现较强细胞毒性的情况，可以尝试在感染后4小时更换成新鲜的完全培养液。2.感染细胞和荧光观察：按照步骤1获得的条件进行病毒感染实验，在观察到EGFP-p65的绿色荧光后，或者在筛选获得EGFP-p65的稳定表达细胞株后，进行后续实验。附录：1.慢病毒使用安全规范：a.作为一种相对安全的病毒，尽管慢病毒在正常细胞内不能进行复制和扩增，但是慢病毒基因组可以整合到被感染细胞的基因组中，因此仍然具有可能的潜在生物学危险。我们建议使用者在病毒操作前应仔细阅读本规范，并在实验中严格按照本规范的要求进行操作。更为严格的美国CDC的生物安全等级及其操作与防护要求参考附表1，也可以访问如下网页：https://www.cdc.gov/labs/pdf/CDC-BiosafetyMicrobiologicalBiomedicalLaboratories-2020-P.pdf。b.慢病毒操作时应使用相应级别的生物安全柜，不同的慢病毒的生物安全等级会有所不同。如果使用普通超净工作台操作病毒，请不要打开排风机，以尽量避免可能污染病毒的尘埃正面吹向操作人员而被吸入。c.实验操作时必需佩戴一次性帽子、口罩，穿戴实验手套及专门的实验服，避免身体直接接触病毒。手部及面部有开放性创口时，禁止进行病毒操作。d.操作病毒时需小心谨慎，不要产生气雾或飞溅。如果操作时超净工作台或其它器皿上有病毒污染，请立即用70%乙醇或2% SDS溶液擦拭干净，或者采取其它的妥善措施。e.如果需要离心，应使用密封性好的离心管，或用封口膜密封后离心，最好使用专门的离心机。f.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%乙醇清理培养瓶或培养板外壁后到显微镜处观察拍照。离开显微镜实验台之前，用70%乙醇擦洗显微镜实验台。g.所有被病毒污染过的枪头、离心管、培养板(皿、瓶)、培养液、手套等，在丢弃前请用84消毒液或2% SDS浸泡过夜。h.脱掉手套后，用肥皂或洗手液清洗双手。i.病毒飞溅或是含有病毒的气溶胶与人体接触，须用大量清水冲洗眼睛、皮肤或粘膜等接触的部位至少15min。j.含病毒的针头或是其它利器刺破皮肤，伤口应立即用10%的碘伏溶液擦洗数分钟，然后用大量清水冲洗。k.装盛慢病毒的实验用品需单独放置，并须加以适当标示。l.对同实验室的人员须进行慢病毒安全培训或安全警示。2.生物安全等级及其操作与防护要求：Summary of Recommended Biosafety Levels for Infectious Agents BSL Agents Practices Primary Barriers andSafety Equipment Facilities(Secondary Barriers) 1 Not known to consistently cause diseases in healthy adults Standard microbiological practices ■ No primary barriers required. ■ PPE: laboratory coats and gloves; eye, face protection, as needed Laboratory bench and sink required 2 ■Agents associated with human disease ■ Routes of transmission include percutaneous injury, ingestion, mucous membrane exposure BSL-1 practice plus: ■ Limited access ■ Biohazard warning signs ■ “Sharps” precautions ■ Biosafety manual defining any needed waste decontamination or medical surveillance policies Primary barriers: ■ BSCs or other physical containment devices used for all manipulations of agents that cause splashes or aerosols of infectious materials ■ PPE: Laboratory coats, gloves, face and eye protection, as needed BSL-1 plus: ■ Autoclave available 3 Indigenous or exotic agents that may cause serious or potentially lethal disease through the inhalation route of exposure BSL-2 practice plus: ■ Controlled access ■ Decontamination of all waste ■ Decontamination of laboratory clothing before laundering Primary barriers: ■ BSCs or other physical containment devices used for all open manipulations of agents ■ PPE: Protective laboratory clothing, gloves, face, eye and respiratory protection, as needed BSL-2 plus: ■ Physical separation from access corridors ■ Self-closing, double-door access ■ Exhausted air not recirculated ■ Negative airflow into laboratory ■ Entry through airlock or anteroom ■ Hand washing sink near laboratory exit 4 ■Dangerous/exotic agents which post high individual risk of aerosol-transmitted laboratory infections that are frequently fatal, for which there are no vaccines or treatments ■ Agents with a close or identical antigenic relationship to an agent requiring BSL-4 until data are available to redesignate the level ■ Related agents with unknown risk of transmission BSL-3 practices plus: ■ Clothing change before entering ■ Shower on exit ■ All material decontaminated on exit from facility Primary barriers: ■ All procedures conducted in Class III BSCs or Class I or II BSCs in combination with full-body, air-supplied, positive pressure suit BSL-3 plus: ■ Separate building or isolated zone ■ Dedicated supply and exhaust, vacuum, and decontamination systems ■ Other requirements outlined in the text BSL, biosafety level; PPE, personal protective equipment.参考文献：1.Liu, T., Zhang, L., Joo, D. et al. Sig Transduct Target Ther. 2017. 2:e17023.2.Giridharan S, Srinivasan M. J Inflamm Res. 2018. 11:407-419. Lenti-CMV-EGFP-p65-EF1α-Puro, i.e., Lentivirus expressing EGFP-p65 fusion protein and puromycin, is a recombinant lentivirus developed by aladdin, which can be used in most mammalian cells (including primary cells and stem cells) to express the EGFP-p65 fusion protein driven by the CMV constitutive promoter. The stable infected cells can be selected with puromycin. This product is mainly used to detect the activation and inhibition of NF-κB signaling pathway, and to observe the nuclear translocation of NF-κB by fluorescence microscopy, etc.NF-κB (Nuclear factor kappa-light-chain-enhancer of activated B cells) is an important nuclear transcription factor involved in cellular response to external stimuli, inflammatory response, immune response and other processes, and is present in almost all types of animal cells. NF-κΒ consists of five subunits Precautions ： Repeated freezing and thawing will reduce the viral titer. If necessary, please store in aliquots after receiving this product. Aliquots must be performed on ice. This product can be stored at 4ºC if it is used within a week, but it should be noted that the viral titer decreases over the time of storage at 4ºC. If stored at -80ºC for more than one year, the titer may decrease, and it is recommended to re-titrate this product.Please read Appendix 1 "Safety Specifications for Lentivirus Use" carefully before using this product. The biosafety level of this product is Biosafety Level 2 (BSL-2). Besides following the standard microbiological practices, it is also necessary to pay attention to limiting exposure, biohazard reminders, prominent warning signs, and develop appropriate safety regulations.Effective protections should be taken during virus operation, and any direct skin exposure is not allowed. Please wash your hands immediately after the experiment. It is strictly forbidden to directly contact the virus. In case of accidental contact, please rinse with water immediately, and properly disinfect the skin with 70% ethanol.Any materials, reagents, and samples that have come into contact with the virus should be disinfected by soaking in 1% SDS solution or disinfectant for more than 30 minutes, or sterilized by autoclaving at 121ºC for 30 minutes.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation. Instructions for Use ： 1.Determination of infection conditions:MOI (Multiplicity of Infection) definition: the ratio of the number of viruses to the number of cells when the virus infects cells. TU (Transduction Units) Definition: The number of biologically active viral particles. After infecting cells with serial dilutions of the virus, the number of biologically active virus particles was determined based on the number of fluorescent cells or the qPCR assay of extracted cellular genomic DNA. The MOI values required for different types of cells are different, and for the first use of lentivirus, it is necessary to optimize the infection conditions. In theory, the higher the MOI value, the higher the infection efficiency, bu with greater cytotoxicity.Therefore, the purpose of the pre-experiment is to determine the MOI value that can make the infection efficiency reach a level suitable for observation while ensuring the cell survival rate.The following protocol is provided for HEK293T cells cultured in 24-well plate.For cells cultured in other type of vessels, the following protocol can be adjusted appropriately.a.One day before infection, inoculate 5×104 cells in 0.5ml of complete culture medium per well of a 24-well plate. The cell density should be about 70-80% at the time of virus infection the next day. Note: The specific cell number of inoculations depends on cell type, cell size, cell growth rate and other factors.b.Calculate the required amount of virus to yield the MOI values of 1, 2, 5, 10, and 20, respectively. The calculation formula is as follows:TU = number of cells at infection* × MOIVirus stock solution (μl) = TU / titer (TU/ml) × 1000*Generally, the number of cells on the second day is calculated by doubling the number of inoculated cells, and the cells that proliferate slowly or do not proliferate can be adjusted appropriately.For example: If 20 MOI of virus are needed for infecting 1 × 105 HEK293T cells, the required TU is 2 × 106TU (1 × 105 × 20)。For a virus stock with a titer of 1×109 TU/ml, the volume of the virus stock will be 2×106 TU / (1×109 TU/ml) × 1000 = 2μl.Note: For a preliminary test of MOI settings for other cell lines, please refer to the table “MOI values recommended by for lentivirus infection of different types of in vitro cultured cells” in the Product Introduction section of this manual or related literaturec.Set up the preliminary test groups according to the figure below. It is recommended to run in duplicate to ensure the accuracy of the experiment. Note: For cells that can be treated with polybrene (, C0351/ST1380), replace the old culture medium with 250μl of fresh culture medium containing 6-8μg/ml polybrene per well; for cells not suitable for treatment with polybrene, discard the old medium and add 250μl of fresh medium to each well. The use of polybrene can increase the infection efficiency by about 2-10 times, and the viability of the virus is determined using polybrene. At this step, fresh culture medium should be added as less as possible to improve the infection efficiency subsequently. Figure 2. Grouping of preliminary tests to determine the optimal MOI value for cell infection. The MOIs were set to be 1, 2, 5, 10, 20, as indicated. Experimental groups with or without 6-8μg/ml polybrene and their corresponding blank groups without virus infection were designed. Blank groups were used to check the cell growth state. The experimental settings in this figure are for reference only, and can be adjusted appropriately based on the requirements of users.d.Thaw this product on ice, mix well, and pipette appropriate amount of virus, calculated in step b, into appropriate wells. Mix gently and continue to culture cells. Note: When the needed amount of virus stock solution is too small, dilute the virus stock solution appropriately with culture medium before adding to platese.At 4 hours postinfection (hpi), add 250μl of fresh culture medium to each well.f.At 24 hpi, replace the virus-containing medium with fresh complete medium to each well, and continue to culture cells. Note: The time for changing culture medium depends on the growth state of cells. If the lentivirus is toxic to cells obviously and affects their growth, the medium should be replaced at least 4 hours after the virus is added.g.After culturing for another 48-96 hours, check the cell growth status and EGFP expression. The optimal MOI value and post-infection time are those under which cell growth is relatively good, with strong EGFP expression and high infection efficiency to facilitate the examination of EGFP.h.If necessary, an appropriate concentration of puromycin (, ST551) can be added to select for infected cells, and then the monoclonal cell line can be obtained by dilution method. The concentration of puromycin can be determined by preliminary tests of serial dilutions of puromycin, and the concentration that can just kill the target cells completely is considered to be appropriate.Note 1:TThe infection efficiency is not necessarily the higher the better for luciferase assay, because too high of infection efficiency could cause cytotoxicity. The infection efficiency of 20-70% is generally sufficient.Note 2: EGFP expression can be examined after 72 hours of lentivirus infection, and there is usually a stronger EGFP expression at about 96 hpi.Note 3: In the case of strong cytotoxicity after infection with lentivirus, replacing the virus-containing medium with fresh complete culture medium at 4 hpi can be attempted.2.Infection of cells and fluorescence observation:Perform the virus infection as described in step 1. After the green fluorescence of EGFP-p65 is visible, or after obtaining the cell lines stably expressing the EGFP-p65, subsequent experiments can be performed. Appendix: 1.Safety specifications for using of lentivirus:a.As a relatively safe virus, although lentivirus cannot replicate and proliferate in normal cells, the lentivirus genome can integrate into the genome of infected cells, so it still has possible potential biological dangers. We suggest that users should read the specifications carefully before virus operation, and operate in strict accordance with the requirements of the specifications. For more stringent US CDC biosafety levels and their operation and protection requirements, please refer to Appendix 1, or visit the following webpage: https://www.cdc.gov/labs/pdf/CDC-BiosafetyMicrobiologicalBiomedicalLaboratories-2020-P.pdf.b.Biosafety cabinets of corresponding levels should be used for lentivirus operations, and the biosafety levels of different lentiviruses will vary. When using an ordinary laminar hood to operate the virus, please do not turn on the exhaust fan to avoid the dust that may be virus-contaminated being blown to the operator and inhaled.c.Disposable hats, masks, laboratory gloves and special laboratory coats must be worn during the experiments to avoid direct contact with the virus. Virus manipulation is prohibited when there are open wounds on the hands and face.d.Be careful not to produce aerosol or splash when handling the virus. If the laminar hood or other utensils are contaminated by the virus during operation, please clean them with 70% ethanol or 2% SDS solution immediately, or take other appropriate measures.e.If centrifugation is required, use a well-sealed centrifuge tube, or seal it with parafilm and centrifuge, preferably using a centrifuge designated for virus operation.f.The following steps should be followed when observing cell infection with a microscope: Tighten the culture flask or cover the culture plate, clean the outer surface of the culture flask or culture plate with 70% ethanol, and then examine by microscope. After finish examining, clean the microscope bench with 70% ethanol.g.All virus-contaminated pipette tips, centrifuge tubes, culture plates (dishes, bottles), culture solution, gloves, and other consumables should be soaked in disinfectant or 2% SDS overnight before discarding.h.After removing gloves, wash hands with soap or hand sanitizer.i.When virus splashes or virus-containing aerosols contact with eyes, skin or mucous membranes, immediately wash with plenty of water for at least 15 minutes.j.If needles or other sharp instruments containing virus pierce the skin, the wound should be immediately scrubbed with 10% iodophor solution for several minutes, and then rinsed with plenty of water.k.The experimental supplies containing lentiviruses should be kept separately and properly marked.l.Lentivirus safety training or safety warnings must be given to personnel in the same laboratory.2.Biosafety levels and their operation and protection requirements:Summary of Recommended Biosafety Levels for Infectious Agents BSL Agents Practices Primary Barriers andSafety Equipment Facilities(Secondary Barriers) 1 Not known to consistently cause diseases in healthy adults Standard microbiological practices ■ No primary barriers required. ■ PPE: laboratory coats and gloves; eye, face protection, as needed Laboratory bench and sink required 2 ■Agents associated with human disease ■ Routes of transmission include percutaneous injury, ingestion, mucous membrane exposure BSL-1 practice plus: ■ Limited access ■ Biohazard warning signs ■ “Sharps” precautions ■ Biosafety manual defining any needed waste decontamination or medical surveillance policies Primary barriers: ■ BSCs or other physical containment devices used for all manipulations of agents that cause splashes or aerosols of infectious materials ■ PPE: Laboratory coats, gloves, face and eye protection, as needed BSL-1 plus: ■ Autoclave available 3 Indigenous or exotic agents that may cause serious or potentially lethal disease through the inhalation route of exposure BSL-2 practice plus: ■ Controlled access ■ Decontamination of all waste ■ Decontamination of laboratory clothing before laundering Primary barriers: ■ BSCs or other physical containment devices used for all open manipulations of agents ■ PPE: Protective laboratory clothing, gloves, face, eye and respiratory protection, as needed BSL-2 plus: ■ Physical separation from access corridors ■ Self-closing, double-door access ■ Exhausted air not recirculated ■ Negative airflow into laboratory ■ Entry through airlock or anteroom ■ Hand washing sink near laboratory exit 4 ■Dangerous/exotic agents which post high individual risk of aerosol-transmitted laboratory infections that are frequently fatal, for which there are no vaccines or treatments ■ Agents with a close or identical antigenic relationship to an agent requiring BSL-4 until data are available to redesignate the level ■ Related agents with unknown risk of transmission BSL-3 practices plus: ■ Clothing change before entering ■ Shower on exit ■ All material decontaminated on exit from facility Primary barriers: ■ All procedures conducted in Class III BSCs or Class I or II BSCs in combination with full-body, air-supplied, positive pressure suit BSL-3 plus: ■ Separate building or isolated zone ■ Dedicated supply and exhaust, vacuum, and decontamination systems ■ Other requirements outlined in the text BSL, biosafety level; PPE, personal protective equipment.References:1.Liu, T., Zhang, L., Joo, D. et al. Sig Transduct Target Ther. 2017. 2:e17023.2.Giridharan S, Srinivasan M. J Inflamm Res. 2018. 11:407-419.

No numbering system information yet

No physical and chemical property information yet

No safety information yet

No production method and use information yet

No upstream information yet

No downstream information yet

No related product information yet