GenTarget's EcoTMPlasmid DNA Miniprep Kit User Manual
Contents
1. Pre made expression Adenovirus product manual Amounts Product Name Catalog AVPOO1 RFP adenovirus AVP002 CFP adenovirus 1x10 IFU ml x 200ul in DMEM with 10 FBS AVP004 Luciferase GFP adenovirus AVPOO5 Luciferase RFP adenovirus AVP Null CMV Null control adenovirus AVPOO1 PBS RFP adenovirus in vivo ready AVP002 PBS CFP adenovirus in vivo ready Luciferase GFP adenovirus in vivo ready Luciferase RFP adenovirus in vivo ready CMV Null control adenovirus in vivo ready 1x10 IFU ml x 200ul in PBS with 5 sucrose AVP004 PBS AVPOO5 PBS AVP Null PBS Storage lt 70 C avoid repeat freeze thaw cycles Stable for 12 months Product Description Adenoviruses are double stranded DNA viruses They can infect a broad range of cell types including dividing and non dividing cells Thus they are used as gene delivery vehicle Amsbio s Adenovirus expression vector is derived from human adenovirus type5 with the E1 and E3 genomic region deleted E1 is essential for the assembly of the virus particles As a result this adenovirus system only produces the replication incompetent adenovirus The viral replication only occurs ina cell line that provides the complementing E1 region such as the 293A cell line where the recombinant adenovirus are produced or amplified Unlike the lentivirus adenoviral construct does not integrate in2. The Null control adenovirus is produced from the adenovector cloned with a Null sequence that does not express any target The AVP Null control virus sold as separate product serves as the negative controls for adenovirus treatment 2 How do know the Adenovirus is working in my cell type The adenovirus can transduce a majority of mammalian cells dividing or non dividing cells like human mouse and other species very well However there are few cell types can not be transduced or with very low efficiency if so you may try to use lentivirus The good way to find out your cell type s susceptibility to adenovirus is to use a fluorescent control adenovirus by which you can easily visualize the fluorescent signal post transduction Amsbio provides pre made adenovirus expressing different fluorescent makers GFP RFP YFP CFP and fluorescent fusion markers which can be used to test the transduction rate for your desired cell type 3 When the target expression occurs and how long it last Typically the target expression can be detected after 12 24 hours Depending on the cell types or dividing cycles the expression peaks at 2 3 days post transduction and persist for as long as the viral genome is present from 1 week to 6 months or longer 4 What s the buffer the virus is provided in Adenovirus is provided as 200ul aliquot in two formats 1 in crude viral stock in DMEM medium with 10 serum or 2 purified adenovirus via column base
3. below for the core expression cassette Adenovirus expression scheme Target OR Target F2A Note Amsbio also provides adenovirus services for cloning of your gene of interest and generates ready to use adenovirus particles with the best prices and fastest turnaround time Key features 1 Pre made ready to use no cloning needed no virus production Simply add the virus to your cell line of interest for target expression 2 Safe to use the adenovirus is produced from the adenovirus vector that has the E1 and E3 gene deleted that are required to generate adenovirus thus the adenovirus are replication incompetent viruses 3 High expression level the target expression is driven by a super strong suCMV promoter with virus titer at 1x10 pfu ml crude virus or 1x10 pfu ml concentrated virus 4 Efficient delivery the adenovirus can transduce actively dividing and non dividing mammalian cells in culture or in vivo and obtain close to 100 expression delivery in most cell types Transduction Protocols 1 Plate your mammalian cells of choice in complete media in appropriate culture conditions and grow overnight 2 The next day cell density should be around 50 75 confluent Thaw the adenoviral stock at room temperature and dilute virus if necessary in PBS or in complete medium or make aliquots on the first use to save virus for next time use directly add the appropriate amount of virus at a suitable MOI i
4. competent cells to directly insert a target into the E1 E3 deleted human adenovirus 5 genome Adenovirus is not integrated into host cell genome therefore it is not suitable for stable long term expression For that reason we do not include any antibiotic marker in the adenovirus constructs In some cases we have included a fluorescent marker under the same promoter for the specific target mediated by 2A element The fluorescent protein provides a marker for monitoring the virus performance and target expression as well as a method of selection via fluorescent signal For long term stable expression please use our pre made lentivirus for which we have included all kind of selection markers 9 Adenovirus vs Lentivirus Both adenovirus and lentivirus can transduce the dividing and non dividing cells Both are used as expression delivery tool for mammalian cell lines or primary cells Unlike the lentivirus adenoviral construct does not integrate into the host genome therefore it does not activate or inactivate host genes For adenovirus the transgene expression decreases gradually as cells divide Therefore Adenovirus delivers transient expression whereas the lentivirus delivers stable long term expression Lentivirus has less immunogenicity to human cells and less toxicity than adenovirus but adenovirus may have better transduction efficiency in most some cell types North America Deutschland Switzerland UK a
5. d system and re suspended into PBS solution with 5 sucrose for in vivo ready status 5 Can pre made adenovirus be used for in vivo delivery applications Yes Amsbio provides adenovirus in PBS with 5 sucrose that is the best solution for direct in vivo applications Please be advised all pre made Adenoviruses are for research use only not for clinical or therapeutic applications 6 What about the bio safety consideration What is RCA Adenoviruses are safe to use They are non replicable viral particles in targeting cells They are produced from the expression vector derived from human adenovirus type5 genome in which the entire E1 4 3kb and E3 region was removed E1 protein is essential for the viral replication Without E1 protein the packaged adenovirus cannot be replicated in target cells The presence of the viral genome is transient and will eventually be diluted out as cell division occurs Note In E1 complementing cell lines like 293A cells the adenovirus can be replicated or amplified Despite the adopted safety features recombinant human adenovirus has been classified as biosafety level Il agent and you need BL 2 level facility to work with it It should be noted that cell culture facilities in most institutes are certified as BL 2 level Please refer to CDC and NIH guidelines for more details about adenovirus handling In rare instances the E1 sequence from the virus production cell genome 293A cells is integrated in
6. mp Rest of World amsbio 23591 El Toro Rd Suite 180 S Lake Forest CA 92630 Tal Tel 1 800 987 0985 Fax 1 949 265 7703 Bockenheimer Landstr 17 19 60325 Frankfurt Main Tel 49 0 69 779099 Fax 49 0 69 13376880 Centro Nord Sud 2E CH 6934 Bioggio Lugano Tel 41 0 91 604 55 22 Fax 41 0 91 605 17 85 184 Milton Park Abingdon OX14 4SE Oxon UK Tel 44 0 1235 828 200 Fax 44 0 1235 820 482 WW n AMS Biotechnology
7. nto cell culture Swirl the plate gently to evenly distribute the virus incubate at 37 C overnight 3 On the following day Day 2 remove the medium containing virus and replace with fresh complete culture medium medium change here is optional depending on the virus toxicity on the target cells 4 Harvest the cells if needed on the desired day e g 2 days post transduction and assay for expression of your recombinant protein Safety Precaution Amsbio adenovirus is replication incompetent However please use extra caution when using viral particles Use the adenovirus in Bio safety Il cabinet Wear gloves at all times when handling viral particles Please refer to CDC and NIH s guidelines for more details regarding to safety issues References 1 Update on Adenovirus and its Vectors J Gen Virol 81 2573 2604 2000 Russell W C et al 2 Supernatant Rescue Assay vs Polymerase Chain Reaction for Detection of Wild Type Adenovirus Contaminating Recombinant Adenovirus Stocks J Virol Methods 56 99 107 1996 Dion L D et al 3 Construction and propagation of human adenovirus vectors In Cell Biology A Laboratory Handbook Ed Celis J E Academic Press San Diego pp 500 512 1998 Hitt M et al Warranty and terms This product is for research use only It is warranted to meet its quality as described when used in accordance with its instructions Amsbio disclaims any implied warranty of this product f
8. or particular application In no event shall Amsbio be liable for any incidental or consequential damages in connection with the products Amsbio s sole remedy for breach of this warranty should be at Amsbio s option to replace the products With the purchase of this product a non transferable right is granted to use the purchased amount of the product for non commercial use No right to resell or re produce this product is granted expressly or by implication FAQ for pre made Adenovirus 1 How to use the pre made Adenovirus What s the control virus What is MOI The pre made Adenovirus is provided in ready to use status Simply add certain amount of adenovirus into your mammalian cell line of choice The amount of virus to be added is dependent on your cell types To obtain the desired expression level you need to use a suitable MOI The multiplicity of infection MOI is defined as the number of virus particles per cell You should test a range of MOls from 0 to 1000 to determine the MOI to use Normally active dividing cell requires less virus amount lower MOI Note the too high MOI may kill your cells because of the toxicity of the virus or of your expressed target the too low MOI may give low expression levels and low positive transduction rate You should use the minimal MOI that will give 100 positive transduction rate or give you the desired expression level or transduction rate For most cell line MOl 1 10 works great
9. to adenoviral transgene construct via sequence homologous recombination which resulted in a replication competent adenovirus RCA In theory this could occur and most likely could happen in large scale virus amplification Wild type replication competent adenoviruses could cause cold symptoms but generally do not cause serious illness We have not observed wild type RCA contamination in small scale All Amsbio adenoviruses are made in very small scale and RCA test is not a routine test for pre made adenovirus For large scale adenovirus application we measure the RCA by PCR tests Note When RCA occurs it will quickly take over the non replicable virus and amplified virus causes cell death To avoid the occurrence of RCA viruses should be produced and amplified in low passage 293 cells 7 How is the virus titer measured The titers Ifu ml of Amsbio s adenovirus are measured via a fluorescent marker GFP or RFP after the virus is transduced into HeLa cells which are also used as titer reference for the same construct backbone containing a non fluorescent target The measured titer is close to the real infectious units that can be measured by a biological assay the plaque assay counting the PFU plaque formation unit after virus transduction 8 How are the adenovirus vector constructed Are there any antibiotic markers included in the adenovirus constructs Amsbio uses its proprietary Eco cloning technology the vector built in cloning
10. to the host genome non insertion virus therefore it does not activate or inactivate host genes Once transduced into the desired mammalian cells the recombinant target will be expressed until the viral genome is diluted out via cell division The transgene expression decreases gradually in active dividing cells normally in 1 2 weeks but the expression can persist for longer time in non dividing or low dividing cells like skeletal muscle cell or neurons as long as the viral genome is still present So adenovirus is a transient expression delivery vehicle Deutschland North America UK amp Rest of World Switzerland amsbio Centro Nord Sud 2E CH 6934 Bioggio Lugano Tel 41 0 91 604 55 22 Fax 41 0 91 605 17 85 Bockenheimer Landstr 17 19 60325 Frankfurt Main Tel 49 0 69 779099 Fax 49 0 69 13376880 23591 El Toro Rd Suite 180 e Lake Forest CA 92630 TT Tel 1 800 987 0985 Fax 1 949 265 7703 184 Milton Park Abingdon OX14 4SE Oxon UK Tel 44 0 1235 828 200 Fax 44 0 1235 820 482 L AMS Biotechnology As qd kes el w ams i Amsbio pre made expression adenovirus contains a target expressed under enhanced human cytomegalovirus suCMV promoter When applicable a fluorescent marker GFP or RFP is bicistronically expressed under the same promoter mediated by a 2A element which allows expression of two individual targets Please see map scheme
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