Lenti-EF1α-Luc-T2A-Puro，即Lentivirus expressing firefly luciferase and puromycin，是阿拉丁自行研发的重组慢病毒，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EF1α启动子适度表达组成型萤火虫萤光素酶(firefly luciferase)和嘌呤霉素(puromycin)抗性基因，感染细胞后可以使用嘌呤霉素筛选稳定株。本产品可作阳性对照或者海肾萤光素酶(renilla luciferase)报告基因的内参，也常被用于萤火虫萤光素酶基因标记的肿瘤细胞系的构建及后续用于活体生物发光成像(活体成像) 以便于进行肿瘤细胞的活体示踪。EF1α (EF1a)启动子是一种来源于延伸因子1α (elongation factor 1 alpha, EF1A)基因的强哺乳动物表达启动子，可在多种细胞中稳定驱动其下游基因的组成型表达，也可以用于干细胞、原代细胞、造血细胞等。T2A是来源于明脉扁刺蛾病毒(Thosea asigna virus, TaV)，是2A肽的一种，编码产生一种具有自我加工能力的短肽，能够在翻译后进行自我剪切，可以实现利用一个转录本翻译出多个蛋白质的功能。T2A目前被认为具有最高的“剪切”效率，很多情况下接近100%，所以通常T2A上下游蛋白的表达水平相当，但上游蛋白的C端会添加一些额外的T2A肽段的残基，而下游蛋白的N端将会有额外的脯氨酸。本产品表达的萤火虫萤光素酶和嘌呤霉素抗性基因的正常功能均不受额外的氨基酸影响。萤光素、萤光素酶、萤火虫萤光素酶和海肾萤光素酶也经常被称作荧光素、荧光素酶、萤火虫荧光素酶和海肾荧光素酶。萤火虫萤光素酶是一种分子量约为61kD的蛋白，在ATP、镁离子和氧气存在的条件下，可以催化luciferin氧化成oxyluciferin，在luciferin氧化的过程中，会发出波长为560nm左右的生物萤光(bioluminescence)。生物萤光可以通过化学发光仪(luminometer)或液闪测定仪进行测定。通过萤光素和萤光素酶这一生物发光体系，可以非常灵敏、高效地检测基因的表达。本病毒中的萤火虫萤光素酶编码进行了改进，确保能更好地在哺乳动物细胞中进行表达。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。阿拉丁的Lenti-EF1α-Luc-T2A-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EF1α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。本产品的滴度不低于10^8TU/ml，适合细胞实验或活体动物实验。TU, transduction unit，即转导单位。TU通过本产品感染HEK293T细胞72小时后，抽提细胞基因组DNA进行qPCR测定。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10^8TU/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，1ml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，1ml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**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，用Bright-Lumi™萤火虫萤光素酶报告基因检测试剂盒(RG051/RG052)检测萤火虫萤光素酶表达情况，以不显著影响细胞生长、萤光素酶表达较强且感染效率便于萤光素酶检测的MOI值和感染后时间为**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：检测细胞萤火虫萤光素酶时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萤火虫萤光素酶报告基因检测，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小时即可进行萤火虫萤光素酶报告基因检测，在96小时左右有较强的萤火虫萤光素酶表达效果。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.感染细胞和萤火虫萤光素酶报告基因检测：按照步骤1获得的条件进行病毒感染实验，在慢病毒感染细胞后48-96小时后，或者在筛选获得萤火虫萤光素酶的稳定表达细胞株后，用Bright-Lumi™萤火虫萤光素酶报告基因检测试剂盒(RG005)检测萤火虫萤光素酶表达情况。 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 and Safety 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 SBSL-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.

Lenti-EF1α-Luc-T2A-Puro, i.e., Lentivirus Expressing Firefly Luciferase and Puromycin, is a recombinant lentivirus developed by aladdin, which can be used in most mammalian cells (including primary cells and stem cells) to moderately express firefly luciferase and puromycin driven by the EF1α constitutive promoter. The stable infected cells can be selected with puromycin.This product can be used as a positive control or an internal reference for the renilla luciferase reporter gene, and is often used for the construction of firefly luciferase gene-labeled tumor cell lines and subsequent in vivo bioluminescence imaging to facilitate in vivo tracking of tumor cells.The EF1α (EF1a) promoter is a strong mammalian expression promoter derived from the elongation factor 1 alpha (EF1A) gene, which can stably drive the constitutive expression of its downstream genes in a variety of cells, including stem cells, primary cells, hematopoietic cells, etc.T2A is a 2A peptide derived from Thosea asigna virus (TaV). It encodes a short peptide with self-cleaving ability after translation to produce multiple proteins from a single transcript. T2A is currently considered to have the highest cleaving efficiency, which is close to 100% in many cases. The expression levels of upstream and downstream proteins of T2A are similar, but some residues of the T2A peptide will be added to the C-terminus of upstream proteins, while downstream proteins will have additional prolines at the N-terminus. The functions of firefly luciferase and puromycin resistance protein expressed using this product are not affected by thse extra amino acids introduced by T2A.Firefly luciferase is a 61kDa monomer that catalyzes the oxidation of luciferin into oxyluciferin in the presence of ATP, Mg2+ and oxygen, generating the chemiluminescence that can be measured by a luminometer or a liquid scintillation counter. This bioluminescence system has been widely used for detecting gene expressions sensitively and efficiently. The firefly luciferase encoding region in this Lentivirus has been optimized to ensure better expression in mammalian cells.This product can infect almost all mammalian cells. After cell infection, the lentivirus will randomly integrate the target genes into the genomic DNA to enable a stable expression of target proteins in infected cells, even in non-dividing cells. Therefore, this product has been widely used in cell and animal experiments.Aladdin's Lenti-EF1α-Luc-T2A-Puro is a replication-deficient lentivirus. The enhancer function of its 3' LTR is lost, forming a self-inactivating 3' LTR, and the U3 region in the 5' LRT is replaced with the EF1α promoter, which cannot replicate and proliferate after infecting ordinary cells, thereby effectively reducing the risk of this product in living organisms. The multiplicity of infection (MOI) values recommended by aladdin for lentivirus infection of different types of in vitro cultured cells are shown in the table below. Cell line Tissue Cancer/cell type Species MOI A431 Epithelial Carcinoma Human 5 A549 Lung Carcinoma Human 5 Astrocytes Nervous system Primary Human 1 B16-F10 Epithelial Melanoma, metastatic Mouse 5 BMM Bone Marrow Primary Human 8 BxPC-3 Pancreas, epithelial Adenocarcinoma Human 10 H3255 Lung Carcinoma, NSCLC Human 10 HCT116 Colon Carcinoma Human 5 HeLa Cervix Carcinoma, epithelioid Human 3 HEK293T Kidney Tumor Human 5 Hepa1-6 Liver Carcinoma Mouse 3 HMVEC Endothelial Endothelial, microvascular Human 100 HT-29 Colon Adenocarcinoma Human 3 HUVEC Umbilicus Endothelial cells Human 100 Jurkat Blood Leukemia, Acute T cell Human 10 LLC-1 Lung Carcinoma Mouse 6 LNCaP Prostate Carcinoma Human 5 MM200 Skin Melanoma Human 5 MCF-7 Breast Adenocarcinoma Human 2 MDA-MB-231 Breast Adenocarcinoma Human 1 MM-AN Skin Melanoma, metastatic Human 16 MMC Breast Carcinoma Mouse 4 MRC-5 Lung, embryonic Fibroblasts Human 1 NB4 Blood Leukemia, acute promyelocytic Human 10 PC12 Adrenal gland Pheochromocytoma Rat 20 SKOV-3 Ovary Adenocarcinoma Human 15 U-2 OS Bone Osteosarcoma Human 5 Comparisons among Retrovirus, Lentivirus, adeno-associated virus and Adenovirus commonly used in laboratories are shown in the table below. Some characteristics of special viruses may differ from those in the table below. Characteristics Retrovirus Lentivirus AAV Adenovirus Genome ssRNA(+) ssRNA(+) ssDNA dsDNA Coat Enveloped Enveloped Naked Naked Particle size 90-100nm 90-100nm 20-30nm 60-90nm Genome size 7-10kb 9kb 5kb 38-39kb Genome integration Yes Yes No No Packaging capacity 2.5-5kb 2.5-6kb 2.5-4.5kb 3-8kb Infection tropism Dividing cells Dividing and non-dividing cells Dividing and non-dividing cells Dividing and non-dividing cells Relative Transduction Efficiency ND 70% 70% 100% Expression started 48-72h 48-72h 72-96h 24-48h Expression duration ＞ 2 months ＞ 2 months ＞ 6 months 3-4 weeks Expression level Medium Medium Medium High Immune response Low Low Very low High In vivo safety Medium Medium High Low Titer before concentration (IFU/ml) 106 107 1011 107 Titer after concentration (IFU/ml) ND 108 0.5-1×1013 1010 Able to obtain high MOI No (≤ 10 copies integrated) No (≤ 10 copies integrated) Yes Yes Biosafety level BSL-2 BSL-2 BSL-1 BSL-2 The titer of this product is no less than 10^8 transduction unit (TU)/ml, which is suitable for cell or live animal experiments. The TU is determined by the qPCR analysis of genomic DNA extracted from HEK293T cells after 72 hours of infection with this product. Multiplicity of Infection (MOI) is the ratio of the number of viruses to the number of cells when the virus infects cells. When 500,000 cells per well of 6-well plates are infected at 5 MOI, 1 ml of this product can infect 40 wells in total; when 100,000 cells per well of 24-well plates are infected at 5 MOI, 1ml of this product can infect 200 wells. The MOI value can be adjusted and the number of wells that can be infected will change correspondingly.