AAV-mCherry-GFP-LC3B (AAV9)

Item Number

A743522

• Product Name: AAV-mCherry-GFP-LC3B (AAV9)
• Specifications & Purity: 10^13vp/ml

Product Description

AAV-mCherry-GFP-LC3B is an AAV9 serotype adeno-associated virus that can express mCherry-GFP-LC3B fusion protein in infected cells or tissues. It can be used for autophagy assays after injection into animals such as mice.Autophagy is a highly conserved intracellular process that degrades long-lived proteins, organelles, and other cytoplasmic components via the lysosomal pathway. Under unfavorable conditions such as starvation, cells degrade excess or abnormal intracellular components through autophagy to provide energy and raw materials for cell survival, promoting growth and development of organisms, cell differentiation, and responses to environmental changes. Abnormal autophagy is closely related to a variety of pathological processes such as tumors, neurodegenerative diseases, metabolic diseases, and pathogen infections. Because of the importance of autophagy in both physiological and pathological processes, it has become a new hot research topic in the field of cell biology.LC3 is the mammalian homolog of ATG8 which is a key protein in yeast autophagy. The LC3 protein family includes LC3A, LC3B, LC3C and GABARAP subfamilies, among which LC3B is the most extensively studied. LC3B has been considered to be the most critical marker protein in the autophagy signaling pathway. The pro-form of LC3B consisting of 125 amino acids is cleaved by Atg4 into the LC3B-I form with 22 amino acids lost at the C-terminus, thus exposing a C-terminal glycine. In the process of autophagy, LC3B-1 undergoes a ubiquitination-like process and becomes the LC3B-II form with phosphatidylethanolamine (PE) covalently linked with the C-terminal glycine, during which ATG7 acts as the E1-like activating enzyme, ATG3 as the E2-like conjugation enzyme, and the Atg12-Atg5-Atg16 complex as the E3-like ligase. Unlike LC3B-I which is localized in cytoplasm, LC3B-II is localized on the inner and the outer membranes of autophagosome. After autophagosome-lysosome fusion, LC3B-II on the outer membrane of autophagosome is cleaved by Atg4, while LC3B-II on the inner membrane is degraded by proteases in lysosome. Although LC3B-II has a larger molecular weight than LC3B-I, it migrates faster than LC3B-I during SDS-PAGE electrophoresis due to its extreme hydrophobicity, appearing 14kD and 16kD, respectively.AAV-mCherry-GFP-LC3B is a recombinant adeno-associated virus developed by aladdin, which can effectively express LC3B proteins fused with red fluorescent mCherry as well as green fluorescent GFP in target cells.In the process of autophagosome-lysosome fusion, the fluorescence of GFP will be quenched due to the acidic environment within lysosome, making it difficult to track the intracellular location of GFP-LC3B. However, mCherry is a monomeric fluorescent protein from mushroom coral and its red fluorescence is stable under acidic conditions. Therefore, the mCherry-EGFP-LC3B fusion protein enables effective tracking of autophagy processes. After infecting cells with the Ad-mCherry-GFP-LC3B adenovirus, non-autophagy cells exhibits diffusive yellow fluorescence in cytoplasm due to the combination of green and yellow fluorescence, while in cells with autophagy, mCherry-GFP-LC3B aggregates on the autophagosome membrane and appears as yellow spots (LC3B dot or punctae) or as red spots due to partial quenching of GFP fluorescence during the process of autophagosome-lysosome fusion.AAV usually does not integrate the target gene into genomic DNA of infected cells, but can stably express target proteins over time, especially in non-dividing cells. AAV also has the advantages of high titer, no significant immune response to in vivo injection, and high efficiency in infecting proliferating and non-proliferating cells.AAV-mCherry-GFP-LC3B is replication-deficient, which can only replicate and amplify in the presence of auxiliary viruses, thus effectively reducing the risk of this product in living organisms.There are mainly 12 serotypes of AAV . Different serotypes of AAV have different coat proteins recognized by different receptors on the cell membrane of infected cells. The tissue affinities of different serotypes of AAV are listed below.AAV SerotypeTissue TropismLiverMuscleBrainRetinaLungHeartPancreasKidneyAAV1√Neuron & glia√√√AAV2√√√AAV3√√√AAV4√√AAV5Neuron & glia√alveolar cellsAAV6√√√AAV7√Neuron√AAV8√√Neuron√√AAV9√√Neuron√√√√√This product is the most widely used AAV9 serotype that can infect a variety of tissues.The transduction efficiency of different cell lines in vitro by different AAV serotypes is listed below . As shown in the table, this product has a very low efficiency in transducting cultured cells and is not suitable for infecting cultured cells. Cell lineAAV1AAV2AAV3AAV4AAV5AAV6AAV8AAV9Huh-7131002.500.1100.70HEK293251002.50.10.150.70.1Hela310020.16.710.20.1HepG2310016.70.31.750.3NDHep1A201000.210.110.2091117100110.20.1170.1NDCHO100100141.433350101COS33100333.351420.5MeWo10100200.36.71010.2NIH3T3101002.92.90.3100.3NDA5491410020ND0.5100.50.1HT118020100100.10.3330.50.1Monocytes1111100NDND1251429NDNDImmature DC2500100NDND2222857NDNDMature DC2222100NDND3333333NDNDPlease refer to the table below for the comparison and the differences in the main features of retroviruses, lentiviruses, adeno-associated viruses and adenoviruses commonly used in the laboratory. The characteristics of some particular viruses may differ from those in the table below.RetrovirusLentivirusAAVAdenovirusGenomessRNA(+)ssRNA(+)ssDNAdsDNACoatEnvelopedEnvelopedNakedNakedParticle size90-100nm90-100nm20-30nm60-90nmGenome size7-10kb9kb5kb38-39kbGenome integrationYesYesNoNoPackaging capacity2.5-5kb2.5-6kb2.5-4.5kb3-8kbInfection tropismDividing cellsDividing and non-dividing cellsDividing and non-dividing cellsDividing and non-dividing cellsRelative Transduction EfficiencyND0.70.71Expression started48-72h48-72h72-96h24-48hExpression duration＞ 2 months＞ 2 months＞ 6 months3-4 weeksExpression levelMediumMediumMediumHighImmune responseLowLowVery lowHighIn vivo safetyMediumMediumHighLowTiter before concentration (IFU/ml)1061071011107Titer after concentration (IFU/ml)ND1080.5-1x10131010Able to obtain high MOINo (≤ 10 copies integrated)No (≤ 10 copies integrated)YesYesBiosafety levelBSL-2BSL-2BSL-1BSL-2aladdin offers adenovirus and adeno-associated virus for the expression of mCherry-GFP-LC3B fusion proteins in infected cells

Precautions：

Repeated freeze-thaws will reduce the viral titer. If necessary, please store in aliquots at the first use. Aliquoting 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 before use.This product is highly efficient and suitable for in vivo infection of animals such as mice, but is inefficient and less suitable for in vitro infection of cultured cells. For autophagy studies of cultured cells, we recommend using the Ad-mCherry-GFP-LC3B (, C3011).For other serotypes of AAV-mCherry-GFP-LC3B, please contact CRO Department for customization.For some valuable knockout or transgenic animals or model animals with disease, it is recommended to perform preliminary tests with 1-2 wild type mice to determine the optimal injection method and dosage before starting formal experiments with the valuable animals.Please read Appendix 1 "Safety Specifications for Lentivirus Use" carefully before using this product. The biosafety level of this product is Biosafety Level 1 (BSL-1). It is not known to consistently cause diseases in healthy adults and can be handled with standard microbiological practices.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.Select an appropriate AAV serotype for the animal tissue to be infected. The AAV9 serotype is the most widely used and can infect a variety of mouse tissues. Please refer to the tissue affinity table for different serotypes of AAV in this manual. 2.Inject this product into animals by tail vein injection, local injection or other injection methods. The recommended dosage per 8-12 weeks mouse for tail vein injection is 2-5×1011 vp, for intracerebral and intraventricular injections is 1-5×109 vp, for intranasal injections is 1-3×1010 vp, for intravitreal injections is 1-4×109 vp. For the optimal injection dosage for autophagy studies of particular tissues, please refer to relevant literature or optimize through preliminary experiments.3.Expression of mCherry-GFP-LC3B fusion protein can usually be visible 3-4 days after injection. Observation of mCherry-GFP-LC3B fusion proteins can also be performed after a longer period of time to determine the cell autophagy in specific tissues.Appendix:1.Safety specifications for handling adeno-associated virusesa.As a relatively safe virus, although the adenovirus genome does not integrate into the host cell genome or replicate in cells after infection, it still has potential biological risks. We recommend that users read the specifications carefully before virus manipulation and operate in strict accordance with the requirements. For more stringent US CDC biosafety levels and their operation and protection requirements, please refer to Appendix 1 or visit the following website: http://www.cdc.gov/biosafety/publications/bmbl5/BMBL5_sect_IV.pdf.b.Biosafety cabinets of corresponding levels should be used for adenovirus operations, and the biosafety levels vary for different adenoviruses. When using an ordinary laminar flow 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 the laminar flow hood or other utensils are contaminated by the virus during operation, please wipe 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 under 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 finishing examination, 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 the virus is splashed or the aerosol containing the virus comes into contact with human body, flush the eyes, skin or mucous membranes with plenty of water for at least 15 minutes.j.When the skin is punctured by a needle or other sharp object containing the virus, the wound should be immediately sterilized with 10% iodine solution for several minutes and then rinsed with plenty of water.2.Calculation of viral MOIMOI (Multiplicity of Infection) is defined as the ratio of the number of viruses to the number of cells when virus infects cells.Pfu (plaque forming units) defines the number of biologically active virus particles.The required pfu can be calculated according to the following formula.Required pfu = number of cells x MOIFor example: If 2 MOI of virus is needed for infecting 1 × 105 cells, the required pfu = (1 × 105 cells) × (2 MOI) = 2 × 105 pfu. For a virus stock with a titer of 1×108 pfu/ml, the volume of the virus stock required will be 2×105 pfu / (1×108 pfu/ml) × 1000 = 2μl.Table 1. Biosafety levels and their handling and protection requirementsBSLAgentsPracticesPrimary Barriers andSafety EquipmentFacilities(Secondary Barriers)1Not known to consistently cause diseases in healthy adultsStandard microbiological practices■ No primary barriers required.■ PPE: laboratory coats and gloves; eye, face protection, as neededLaboratory bench and sink required2■ Agents associated with human disease■ Routes of transmission include percutaneous injury, ingestion, mucous membrane exposureBSL-1 practice plus: ■ Limited access■ Biohazard warning signs■ “Sharps” precautions■ Biosafety manual defining any needed waste decontamination or medical surveillance policiesPrimary 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 neededBSL-1 plus:■ Autoclave available3Indigenous or exotic agents that may cause serious or potentially lethal disease through the inhalation route of exposureBSL-2 practice plus: ■ Controlled access■ Decontamination of all waste■ Decontamination of laboratory clothing before launderingPrimary 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 neededBSL-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 exit4■ 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 transmissionBSL-3 practices plus:■ Clothing change before entering■ Shower on exit■ All material decontaminated on exit from facilityPrimary barriers:■ All procedures conducted in Class III BSCs or Class I or II BSCs in combination with full-body, air-supplied, positive pressure suitBSL-3 plus:■ Separate building or isolated zone■ Dedicated supply and exhaust, vacuum, and decontamination systems■ Other requirements outlined in the textBSL, biosafety level; PPE, personal protective equipment.Related Products:Cat. No.Product NamePack SizeC3006-1mlAd-GFP-LC3B1mlC3006-10mlAd-GFP-LC3B10mlC3011-1mlAd-mCherry-GFP-LC3B1mlC3011-10mlAd-mCherry-GFP-LC3B10mlC3013-250μlAAV-mCherry-GFP-LC3B 250μlC3015-1mlAd-GFP-p621mlC3015-10mlAd-GFP-p6210mlC3016-1mlAd- mCherry-p621mlC3016-10mlAd- mCherry-p6210mlD2761-1μgpCMV-GFP-LC3B1μgD2761-100μgpCMV-GFP-LC3B100μD2762-1μgpCMV-mCherry-GFP-LC31μgD2762-100μgpCMV-mCherry-GFP-LC3100μgD2763-1μgpCMV-GFP-p621μgD2763-100μgpCMV-GFP-p62100μgD2764-1μgpCMV-mCherry-p621μgD2764-100μgpCMV-mCherry-p62100μg