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1. Product Manual RAPAd CMV Adenoviral Bicistronic Expression System GFP Catalog Number VPK 254 1 kit FOR RESEARCH USE ONLY Not for use in diagnostic procedures CELL BIOLABS INC Creating Solutions for Life Science Research Introduction Recombinant adenoviruses have tremendous potential in both research and therapeutic applications There are numerous advantages they provide when introducing genetic material into host cells The permissive host cell range is very wide The virus has been used to infect many mammalian cell types both replicative and non replicative for high expression of the recombinant protein Recombinant adenoviruses are especially useful for gene transfer and protein expression in cell lines that have low transfection efficiency with liposome After entering cells the virus remains epichromosomal i e does not integrate into the host chromosome so does not activate or inactivate host genes Recently recombinant adenoviruses have been used to deliver RNAi into cells Two methods have traditionally been used to generate recombinant adenoviruses The first involves homologous recombination of a shuttle vector containing gene of interest and an adenoviral backbone plasmid vector restricted in E1 E3 in an adenovirus packaging cell line The isolation of recombinant adenovirus by this method involves performing multiple plaque isolations to avoid wild type virus and is extremely laborious and time consuming The2. TTGGTACCGTT TAAACTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCTGCAGCCCGGGGGATCCACTAGT Cla EcoR V BamH 5 ly CELL BIOLABS INC Pacl Pacl pacAd5 9 2 100 34 9 kb AE3 A720 bp Ad5 Figure 2 pacAd5 9 2 100 Vector 34947 bp Ampicillin resistant The novel pacAd5 9 2 100 Ad backbone vector is devoid of the left hand ITR the packaging signal and E1 sequences Pacl eGFP SV40pA pacAd5 CMV eGFP 6 9 kb Figure 3 pacAdS5 CMV GFP Control Vector 6935 bp Ampicillin resistant pacAd5 CMV GFP Features 3 10 Pacl 16 368 1 353 of Ad5 385 912 CMV Promoter 992 1711 GFP 1713 2160 SV40 pA 2161 4615 3328 5792 of Ad5 5867 6727 B Lactamase CELL BIOLABS INC Pacl pacAd5 CMV ntLacZ Mace 9 4 kb SV40pA Figure 4 pacAd5 CMV ntLacZ Control Vector 9278 bp Ampicillin resistant pacAd5 CMV ntLacZ Features 3 10 16 368 385 912 1105 4148 4193 4640 4641 7095 8347 9210 Pacl 1 353 of Ad5 CMV Promoter ntLacZ SV40 pA 3328 5792 of Ad5 B Lactamase Preparation of Recombinant Adenovirus I Vector Linearization with PaclI 1 Digest a sufficient amount of the pacAdS CMV IRES GFP shuttle vector containing gene of interest and the pacAd5 9 2 100 Ad backbone vector with Pacl 2 Run 0 5 ug of each digested DNA and undigested DNA on a 0 8 agarose gel to confirm the completion of PacI digestion For pacAd5 9 2 100 one band of 33 kb and a second ba
3. second method pAdEasy system employs the homologous recombination machinery in E coli a recombinant adenovirus is produced by a double recombination event between cotransformed adenoviral backbone plasmid vector and a shuttle vector carrying the gene of interest For the pAdEasy method the system is high fidelity but inefficient and requires the screening of many bacterial colonies This results in a significant time commitment even before transfection of recombinant DNA into El expressing cells such as HEK293 cells Cell Biolabs RAPAd Adenoviral Expression System provides a much faster and safer method to generate RCA free recombinant adenovirus at high titer see Table 1 The RAPAd system uses a novel Ad backbone devoid of the left hand ITR the packaging signal and El sequences There is no need to perform the bacterial in vitro homologous recombination pAdEasy method and also the multiple plaque isolations standard homologous recombination method in packaging cell line The RAPAd system allows for generation of a recombinant virus within 2 weeks and the virus produced contained virtually no contaminating Ela sequences or replication competent virus RCA Cell Biolabs RAPAd Adenoviral Expression System is simple to use The method is straightforward and requires very limited hands on time from shuttle backbone cotransfection to the isolation of virus particles It produces equivalent infectious titers as the standard vi
4. CAAGCAGT TCCAGGCGGT CCCACAGCT CGGTCACCTGCTCTACGGCA TCTCGATCCAGCATATCTCCTCGTTTCGCGGGTTGGGGCGGCTTTCGCT GTACGGCAGTAGTCGGTGCT CGTCCAGACGGGCCAGGGTCATGTCTTTCCACGG GCGCAGGGTCCTCGTCAGCGTAGTCTGGGTCACGGTGAAGGGGT GCGCT CCGGGCT GC GCGCTGGCCAGGGTGCGCTTGAGGCTGGTCCTGCTGGTGCTGAAG CGCTGCCGGTCTTCGCCCTGCGCGTCGGCCAGGTAGCATTTGACCATGGTGTCATAGT CCAGCCCCTCCGCGGCGTGGCCCTTGGCGCGCAGCTTGCCCTTGG AGGAGGCGCCGCACGAGGGGCAGTGCAGACTTTTGAGGGCGTAGAGCTT GGGCGCGAGAAATACCGATT CCGGGGAGTAGGCATCCGCGCCGCAGGCCCCGCA GACGGTCTCGCATTCCACGAGCCAGGTGAGCTCTGGCCGTTCGGGGTCAAAAACCAGGTTTCCCCCATGCTTTTTGATGCGTTTCTTACCTCTGGTTTCCATG AGCCGGTGTCCACGCTCGGTGACGAAAAGGCTGTCCGT GTCCCCGTATACAGACT TGAGAGGCCTGTCCTCGACCGATGCCCTTGAGAGCCTTCAACCCAGTC AGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCGCACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTGGGTCA TT TTCGGCGAGGACCGCTTTCGCTGGAGCGCGACGATGATCGGCCTGTCGCTTGCGGTATTCGGAATCT TGCACGCCCTCGCTCAAGCCTTCGTCACTGGTCC CGCCACCAAACGTTTCGGC GAGAAGCAGGCCATTATCGCCGGCATGGCGGCCGACGCGCTGGGCTACGT CTTGCTGGCGTTCGCGACGCGAGGCTGGATGGCC TTCCCCATTATGATTCTTCTCGCTTCCGGCGGCATCGGGATGCCCGCGT TGCAGGCCATGCTGTCCAGGCAGGTAGATGACGACCATCAGGGACAGCTTCAAG GCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAG GTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCC GCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAAC CCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACA
5. CACGACTTCTTCAAG TCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTT CAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGT TCGAGGGCGACACCCTGG TGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGC TGGAGTACAAC TACAACAGCCACAACGTCTATATCATGGC CGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATC GGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGT TCGTGACCGCCGCCGGGAT CACTCTCGGCATGGACGAGCTGTACAAGTAAGCGGCCGCCACCGCGGGGAGATCCAGACATGATAAGATACATTGATGAGTTTG GACAAACCACAACTAGAAT GCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAA CAACAACAATTGCATTCATTTTATGTTTCAGGTT CAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATGAT CCCGGCTGCCTCGCGCGTT TCGGTGATGACGGTGAAAACCTCTTGACACATGCAGCTCCCGGAGACGGT CACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGA CAAGCCCGTCAGGGCGCGT CAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGAGGT CGACTCTAGTCCCCGCGGTGGCAGATCTGGAAGGTGCTGAGGTAC GATGAGACCCGCACCAGGT GCAGACCCTGCGAGT GT GGCGGTAAACATAT TAGGAACCAGCCTGTGATGCTGGAT GTGACCGAGGAGCTGAGGCCCGATCACT TGGTGCTGGCCTGCACCCGCGCTGAGTTTGGCTCTAGCGATGAAGATACAGAT TGAGGTACTGAAAT GT GTGGGCGTGGCTTAAGGGTGGGAAAGAATATATA AGGTGGGGGTCTTATGTAGTTTTGTATCTGTTTTGCAGCAGCCGCCGCCGCCATGAGCACCAACTCGTT TGATGGAAGCATTGTGAGCTCATATTTGACAACG CGCATGCCCCCATGGGCCGGGGT GC GTCAGAATGTGAT GGGCTCCAGCATTGATGGTCGCCCCGTCCTGCCCGCAAACTCTACTACCTTGACCTACGAGACCG TGTCTGGAACGCCGTTGGAGACTGCAGCCTCCGCCGCCGCTTCAGCCGCTGCAGCCACCGCCCGCGGGATTGTGACTGACTTTGCTTTCCTGAGC
6. CATTAACCTATAAAAATAGGCGTATCACGA GGCCCTTTCGTCTTCAAGAA References 1 Bett AJ Haddara W Prevec L and Graham FL 1994 Proc Natl Acad Sci U S A 91 8802 6 Homologous recombination in packaging cell line 2 He T C Zhou S da Costa L T Yu J Kinzler K W et al 1998 Proc Natl Acad Sci USA 95 2509 14 pAdEasy System 3 RD Anderson R E Haskell H Xia B J Roessler and B L Davidson 2000 Gene Ther 7 1034 8 RAPAd System Recent Product Citations 1 Lozi M et al 2015 Overexpression of oxytocin receptors in the hypothalamic PVN increases baroreceptor reflex sensitivity and buffers BP variability in conscious rats Br J Pharmacol 171 4385 4398 2 Brunton P J et al 2015 5a reduced neurosteroids sex dependently reverse central prenatal programming of neuroendocrine stress responses in rats J Neurosci 35 666 677 3 Lozi M et al 2014 Overexpression of oxytocin receptors in the hypothalamic PVN increases baroreceptor reflex sensitivity and buffers BP variability in conscious rats Br J Pharmacol 171 4385 4398 10 CELL BIOLABS INC Notice to Purchaser This product is sold for research and development purposes only and is not to be incorporated into products for resale without written permission from Cell Bio
7. CCGCT TGC AAGCAGTGCAGCTTCCCGT TCATCCGCCCGCGAT GACAAGTTGACGGCT CTT TTGGCACAATTGGATTCTTTGACCCGGGAACTTAATGTCGTTTCTCAGCAG CTGTTGGATCTGCGCCAGCAGGTTTCTGCCCTGAAGGCTTCCTCCCCTCCCAATGCGGTT TAAAACATAAATAAAAAACCAGACTCTGTTTGGATTTGGATCA AGCAAGTGTCTTGCTGTCTTTATTTAGGGGTTTTGCGCGCGCGGTAGGCCCGGGACCAGCGGTCTCGGT CGTTGAGGGTCCTGTGTATTTTTTCCAGGACGTG GTAAAGGTGACTCTGGATGTTCAGATACATGGGCATAAGCCCGTCTCTGGGGTGGAGGTAGCACCACTGCAGAGCTTCATGCTGCGGGGTGGTGTTGTAGATG ATCCAGTCGTAGCAGGAGC GCTGGGCGTGGTGCCTAAAAATGTCTTTCAGTAGCAAGCTGATTGCCAGGGGCAGGCCCTTGGTGTAAGTGTTTACAAAGCGGT d N CELL BIOLABS INC TAAGCTGGGATGGGTGCATACGTGGGGATATGAGAT GCATCTTGGACTGTATTTTTAGGTTGGCTATGT TCCCAGCCATATCCCTCCGGGGATTCATGTTGTG CAGAACCACCAGCACAGTGTATCCGGTGCACTTGGGAAATTTGTCATGTAGCTTAGAAGGAAAT GCGTGGAAGAACTTGGAGACGCCCTTGTGACCTCCAAGA TT TTCCATGCATTCGTCCATAATGATGGCAATGGGCCCACGGGCGGCGGCCTGGGCGAAGATATTTCTGGGATCACTAACGTCATAGTTGTGTTCCAGGATGA GATCGTCATAGGCCATTTT TACAAAGCGCGGGCGGAGGGT GCCAGACTGCGGTATAAT GGT TCCATCCGGCCCAGGGGCGTAGTTACCCTCACAGATTTGCAT TTCCCACGCTTTGAGTTCAGATGGGGGGATCATGTCTACCTGCGGGGCGATGAAGAAAACGGTT TCCGGGGTAGGGGAGATCAGCTGGGAAGAAAGCAGGTTC CTGAGCAGCTGCGACTTACCGCAGCCGGTGGGCCCGTAAATCACACCTATTACCGGGT GCAACTGGTAGT TAAGAGAGCTGCAGCTGCCGTCATCCCTGAGCA GGGGGGCCACTTCGTTAAGCATGTCCCTGACTCGCATGTTTTCCCTGACCAAATCCGCCAGAAGGCGCT CGCCGC CCAGCGATAGCAGT TCT TGCAAGGAAGC AAAGTTTTTCAACGGTTTGAGACCGTCCGCCGTAGGCATGCTTTTGAGCGTTTGAC
8. CGACTTATCGCCACTGGCAGCAGCCACTGGTAACAG GATTAGCAGAGCGAGGTAT GTAGGC GGT GCTACAGAGT TCT TGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTG AAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGT GGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCA GAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACT CACGTTAAGGGATTTTGGTCATGAGATTATCAAA AAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGT GAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCT GACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCA GTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGC CAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTT ATCCGCCTCCATCCAGTCTATTAAT TGTTGCCGGGAAGCTAGAGTAAGT AGT TCGCCAGTTAATAGTTT GCGCAACGTTGTTGCCATTGCTGCAGGCATCGTG GTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGC GAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCT TCGGTCCTCCGATCGTTGT CAGAAGTAAGT TGGCCGCAGTGTTATCACT CATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATG CTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCA CATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGC GAAAACTCTCAAGGATCTTACCGCT GTTGAGATCCAGTTCGATGTAACCCACTC GTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAAT GCCGCAAAAAAGGGAATAAGGGCGACACG GAAATGTTGAATACTCATACTCTTCCTTITTTCAATATTATTGAAGCATT TATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAAT AAACAAATAGGGGTTCCGCGCACAT TTCCCCGAAAAGT GCCACCTGACGTCTAAGAAACCATTATTATCATGA
9. TTACGGTAAATGGCCCG CCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGT ATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGC CCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCAT GGTGATGCGGTTTTGGCAGTACATCAATGGGCGT GGATAGCGGTTTGACTCACGGGGAT TTCCAAGTCTCCACCCCAT TGACGT CAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTA ACAACTCCGCCCCATTGACGCAAAT GGGCGGTAGGCGT GTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGT CAGTTGGTACCGTTTAAACT CGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCTGCAGCCCGGGGGATCC TCGAGGGCCGGCGCGCCGCGGCCGCTACGTAAATTCCGCCCCTCTCCC TAACGTTACTGGCCGAAGC CGCTT GGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAA CCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGT CTTTCCCCTCTCGC CAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAG CTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAAC CCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATAC ACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGT GAGTTGGATAGTTGTGGAAAGAGT CAAATGGCTCTCCTCAAGCGTAT TCAACAAGGGGCTGAAGGAT GCCCAGAAGGTACCCCATT GTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGT GTTTAGTCGAGGT TAAAAAAACGTCTAGGCCCCCCGAACC ACGGGGACGTGGTTTTCCT TTGAAAAACACGATGATAATATGGCCACAAC CATGGT GAGCAAGGGCGAGGAGCT GTTCACCGGGGTGGTGCCCATCCTGGTCG AGCTGGACGGCGACGTAAACGGCCACAAGT TCAGCGTGTCCGGC GAGGGC GAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGG CAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGC GT GCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAG
10. irus Concentration and Purification Kit RAPAd Universal Adenoviral Expression System RAPAd RSV Adenoviral Expression System RAPAd miRNA Adenoviral Expression System Kit Components 1 pacAdS CMV IRES GFP Shuttle Vector Part No 325401 One 40 uL vial at 0 25 mg mL pacAd5 9 2 100 Vector Part No 325002 One 40 uL vial at 0 25 mg mL 2 3 pacAdS CMV GFP Control Vector Part No 325004 One 40 uL vial at 0 25 mg mL 4 pacAd5 CMV ntLacZ Control Vector Part No 325202 One 40 uL vial at 0 25 mg mL Materials Not Supplied 1 293 cells we recommend 293AD Cell Line Cat AD 100 for high titer production of recombinant adenovirus 2 293 Cell Culture Medium 3 Transfection Reagents 4 Pacl New England Biolabs Cat RO547L Storage Upon receipt store all kit components at 20 C until their expiration dates Safety Considerations Remember that you will be working with samples containing infectious virus Follow the recommended NIH guidelines for all materials containing BSL 2 organisms AN CELL BIOLABS INC JE D Vector Features pacAd5 CMV IRES GFP 7 5 kb Figure 1 pacAd5 CMV IRES GFP shuttle Vector 7539 bp Ampicillin resistant pacAd5 CMV IRES GFP Features 3 10 Pacl 16 368 1 353 of Ad5 382 912 CMV Promoter 919 964 MCS 983 1596 IRES 1597 2316 GFP 2317 2764 SV40 pA 2759 5223 3328 5792 of Ad5 6471 7331 B Lactamase Multiple Cloning Sites Pme EcoR
11. labs The patented RAPAd technology is covered by a license from University of Iowa By the use of this product you accept the terms and conditions of all applicable Limited Use Label Licenses You may contact our Business Development department at busdev cellbiolabs com for information on sublicensing this technology Warranty These products are warranted to perform as described in their labeling and in Cell Biolabs literature when used in accordance with their instructions THERE ARE NO WARRANTIES THAT EXTEND BEYOND THIS EXPRESSED WARRANTY AND CELL BIOLABS DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF FITNESS FOR PARTICULAR PURPOSE CELL BIOLABS sole obligation and purchaser s exclusive remedy for breach of this warranty shall be at the option of CELL BIOLABS to repair or replace the products In no event shall CELL BIOLABS be liable for any proximate incidental or consequential damages in connection with the products Contact Information Cell Biolabs Inc 7758 Arjons Drive San Diego CA 92126 Worldwide 1 858 271 6500 USA Toll Free 1 888 CBL 0505 E mail tech cellbiolabs com www cellbiolabs com 2010 2015 Cell Biolabs Inc All rights reserved No part of these works may be reproduced in any form without permissions in writing 11 J _ CELL BIOLABS INC C
12. nd of 2 0 kb 3 Remove buffer and enzyme from the remainder of the restriction reactions by phenol extraction ethanol precipitation or using a similar DNA purification kit 4 Resuspend the DNA in sterile dH20 Store the digested DNA at 20 C II Transfection 1 Seed 2 x 10 cells in a 60 mm culture dish without antibiotics one day before transfection 7 CELL BIOLABS INC A After 16 to 24 hours start transfection when the culture becomes 70 80 confluence Note We suggest transfecting cells with FuGENE Transfection Reagent Roche Applied Science or Lipofectamine Plus Invitrogen For example 4 ug of pacAdS CMVK NpA shuttle vector and I ug of pacAd5 9 2 100 Ad backbone vector are mixed with 9 uL FuGENE Transfection Reagent according to the manufacturer s recommendation The mixed DNA FuGENE complex is added by dropwise into the culture media Aspirate the media containing transfection reagent the next day and add 4 mL of complete culture medium After incubating for 7 days check for the presence of plaques If plate is ready for harvest gt 50 of cells lifted then collect the Crude Viral Lysate If not feed the cells with 1 mL of complete culture medium continue to incubate at 37 C with CO2 On day 10 check for the presence of plaques If plate is ready for harvest gt 50 of cells lifted then collect the Crude Viral Lysate If not feed the cells with 1 mL of complete culture medium c
13. ontinue to incubate at 37 C with CO2 Keep checking plate for the presence of plaques Do not keep plate more than 15 days III Harvesting the Crude Viral Lysate 1 2 3 4 Harvest adenovirus containing cells by squirting cells off the plate with a 5 or 10 mL sterile serological pipette Transfer cells and media to a sterile 15 ml tube Scrape the cells into the medium with a cell lifter if necessary Release viruses from cells by three freeze thaw cycles 10 minutes each in 37 C water bath and dry ice methanol bath Centrifuge the cell lysate in a table top centrifuge at 3000 rpm for 15 minutes at room temperature to pellet the cell debris Aliquot and store the Crude Viral Lysate Initial Viral Stock at 80 C IV Amplification Note The following procedure is suggested for T75 flasks and may be optimized to suit individual needs 1 2 Seed 3 5 x 10 cells in a T75 flask one day before infection Add 50 of the above Crude Viral Lysate to the culture We recommend using a multiplicity of gt 0 5 PFU plaque forming units or enough viruses that cells demonstrate cytopathic effects CPEs within 48 hrs During 24 48 hr infection examine the monolayer twice per day under the microscope for CPE When CPE is nearly complete i e most cells rounded but not yet detached from the flask harvest cells by pipetting media up and down to wash the infected cells from the flask into the media Pool infected cells and medi
14. ral genome shuttle plasmid recombination method In Cell Biolabs RAPAd CMV Adenoviral Bicistronic Expression System GFP the shuttle vector contains a CMV ahead of the multiple cloning sites followed by IRES GFP 2 les CELL BIOLABS INC A _ Standard pAdEasy RAPAd Homologous Expression Expression Recombination System System Cotransfect 293 cells with Linearize Linearize Shuttle Vector and Shuttle Vector and Ad Shuttle Vector RAPAd Ad Backbone Backbone Vector using Pmel Vector using PaclI Multiple Cotransform E coli BJ5183 Cotransfect Plaque cells with linearized Shuttle 293 cells Isolations Vector and pAdEasy Vector Virus Recombinant selection by Viral Stock Amplification restriction enzyme analysis Viral Stock Linearize recombinant plasmid using Pacl Transfect 293 cells Viral Stock 12 18 weeks 8 9 weeks 2 3 weeks Table 1 Outline of Recombinant Adenovirus Systems 3 h IN CELL BIOLABS INC JaA ae Related Products SOS CO St OO ee ee oS AD 100 293AD Cell Line AD 200 ViraDuctin Adenovirus Transduction Reagent VPK 090 VPK 099 VPK 100 VPK 109 VPK 110 VPK 111 VPK 130 10 VPK 250 11 VPK 251 12 VPK 253 ViraBind Lentivirus Concentration and Purification Kit ViraBind Adenovirus Miniprep Kit ViraBind Adenovirus Purification Kit QuickTiter Adenovirus Titer Immunoassay Kit QuickTiter Adenovirus Titer ELISA Kit Rapid RCA Assay Kit ViraBind Retrov
15. um Pellet cells by centrifugation at 1000 g for 5 minutes Remove supernatant resuspend cell pellet in medium or in 10 mM Tris pH 8 0 100 mM NaCl 0 25 0 5 mL per T75 flask Release the adenoviruses from the cell suspension with three freeze thaw cycles Centrifuge at 3000 g for 10 minutes to pellet the cell debris Discard the pellet and save supernatant as viral stock The viral supernatant can be stored at 80 C or immediately purified or titered 8 AN CELL BIOLABS INC N ZA Example of Results The following figures demonstrate typical results of generating recombinant adenovirus One should use the data below for reference only This data should not be used to interpret actual results Figure 5 Generation of recombinant adenovirus using the RAPAd Adenoviral Expression System 293 cells were transfected with Pacl linearized pacAd5 CMV GFP vector and pacAd5 9 2 100 vector Plates were examined for the presence of viral foci under inverted fluorescence microscope Appendix pacAd5 CMV IRES GFP Plasmid Sequence AATTAATTAAGCTAGCATCATCAATAATATACCTTATTTTGGATTGAAGCCAATATGATAATGAGGGGGTGGAGT TTGTGACGTGGCGCGGGGCGTGGGAACG GGGCGGGTGACGTAGTAGT GTGGCGGAAGTGTGATGT TGCAAGTGTGGC GGAACACAT GTAAGCGACGGAT GTGGCAAAAGTGACGTTTTTGGTGTGCGCCGG TGTACACAGGAAGTGACAATTTTCGCGCGGTTTTAGGCGGATGT TGTAGTAAATT TGGGCGTAACCGAGTAAGATTTGGCCATT TTCGCGGGAAAACTGAATA AGAGGAAGTGAAATCTGAATAATTTTGTGTTACTCATAGCGCGTAATAT TTGTCTAGGGAGATCAGCCT GCAGGT CGTTACATAAC
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