Lenti-EF1α-Luc-T2A-Puro

Item Number

L747629

• Specifications & Purity: 10^8TU/ml
• Stability And Storage: Store at -80℃ for up to 1 year, -20℃ for up to 1-2 months, or 4℃ for up to 1 week.
• Storage Temp: Store at -80°C
• Shipped In: Ice chest + Ice pads

Product Description

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.

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 4℃ if it is used within a week, but it should be noted that the viral titer decreases over the time of storage at 4℃. If stored at -80℃ 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). Beside 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 for more than 30 minutes, or sterilized by autoclaving at 121℃ 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 is 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, but with greater cytotoxicity. Therefore, the purpose of the preliminary test 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 × 106 TU (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 Firefly Luciferase Assay Systeim ( Firefly Luciferase Assay System (RG005) to analyze the expression of firefly luciferase. 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 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. 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