ViraSafe™ Universal Lentiviral Expression System, Ecotropic
Contents
1. GTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTA GGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCT TGATCCGGCAAACAAACCACCGCTGGTAGCGGTGG TTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTT CTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGA GGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATG AAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTA CGTTCA TCCATAGTTGCCTGACTCCTGCGCAGTCCAAAAAAAAAGGCTCCAAAAGGAGCC AATTGTATCGGTGGGCCCTTAGAAAAACTCATCGAGCATCAAATGA AACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAA GATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGT GACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACC AAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACA CTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATC AGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTG CCATG CAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATA AATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACG CCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAG TTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGTTTAAACAAATAGTC2. Product Manual ViraSafe Universal Lentiviral Expression System Ecotropic Catalog Number VPK 211 ECO 1 kit FOR RESEARCH USE ONLY Not for use in diagnostic procedures CELL BIOLABS INC Creating Solutions for Life Science Research Introduction Lentivirus vector based on the human immunodeficiency virus 1 HIV 1 has become a promising vector for gene transfer studies The advantageous feature of lentivirus vector is the ability of gene transfer and integration into dividing and non dividing cells The pseudotyped envelope with vesicular stomatitis virus envelope G VSV G protein broadens the target cell range Lentiviral vectors have been shown to deliver genes to neurons lymphocytes and macrophages cell types that previous retrovirus vectors could not be used Lentiviral vectors have also proven to be effective in transducing brain liver muscle and retina in vivo without toxicity or immune responses Recently the lentivirus system is widely used to integrate siRNA efficiently in a wide variety of cell lines and primary cells both in vitro and in vivo Lentivirus particles are produced from 293T cells through transient transfection of plasmids that encode for the components of the virion Figure 1 Due to safety concerns regarding the infectious nature of HIV 1 recent lentiviral packaging systems have separated the viral components into 3 or 4 plasmids However these systems still present a small chance of generating replica
3. also be cloned into the vector optional MCS BamHI EcoRI EcoRV Sall 1073 1100 MCS GGGGGATCCGCGGAATTCGTCGATATCAGCGTCGACAAT BamHI EcoRI EcoRV Sall Figure 2 pSMPUW Lentiviral Expression Vector 4632 bp Kanamycin resistant EcoRI XhoI Digestion 1251 bp 3381 bp Note Bacterial culture of psMPUW vectors should be done in medium containing 10 ug mL Kanamycin For maximal plasmid yield and quality we recommend Stbl3 endoA1 4 competent cells Invitrogen and treatment with alkaline proteinase Promega A1441 or Sigma P8038 for 4 5 min using 10 units of proteinase per mL of bacterial lysate before adding neutralization solution Figure 3 pRSV Rev Packaging Vector 4180 bp Ampicillin resistant EcoRI Digestion 300 bp 3880 bp em BEER Figure 4 pCMV Eco Envelop Vector 6763 bp Ampicillin resistant BamHI Digestion 777 bp 5986 bp Figure 5 pCgpV Packaging Vector 9118 bp Ampicillin resistant Pst I Digestion 927 bp 1424 bp 6767 bp CELL BIOLABS INC e Lentivirus Production 1 2 One day before transfection plate sufficient 293T cells or 293LTV cells Cat LTV 100 to achieve 70 80 confluence on the day of transfection Transfect cells by Calcium Phosphate or other transfection reagents Note We suggest transfecting cells with FuGENE Transfection Reagent Roche Applied Science or Lipofectamine Plus Invitrogen We recommend the ratio of vectors at 3 1 1 1 pSMPUW DCMV
4. AAAAGCCTCCGGCG References 1 Chen M et al 2002 Nature Genetics 32 4 670 675 2 Naldini L U Blomer P Gallay D Ory R Mulligan F H Gage I M Verma and D Trono 1996 Science 272 263 267 Verma I M and N Somia 1997 Nature 389 239 242 4 Kahl C A Marsh J Fyffe J Sanders D A and K Cornetta 2004 J Virol 78 1421 30 5 White S M Renda M Nam N Y Klimatcheva E Zhu Y Fisk J Halterman M Rimel B J Federoff H Pandya S Rosenblatt J D and V Planelles 1999 J Virol 73 2832 40 6 Kafri T van Praag H Ouyang L Gage F H and I M Verma 1999 J Virol 73 576 84 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 Biolabs The patented technology is covered by a license from CHLA and University of Southern California 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
5. Eco pRSV REV pCgpV Harvest lentiviral supernatant 36 72 hours after transfection Supernatant can be harvested 2 or 3 times every 12 hours Keep it at 4 C over the collecting period Pool the collected supernatants centrifuge 5 minutes at 1500 rpm to remove cell debris and filtrate on 0 22 um Supernatants can be used directly or purified concentrated if needed For long term storage store supernatant at 80 C in aliquots Post Packaging Considerations Packaging your lentivirus is only the first step to ensuring successful expression of your gene The following steps should be considered prior to infection of your host cell l Concentration and purification of your lentivirus Because of the latent nature of lentivirus it is imperative that your virus be highly concentrated before infecting your host cell Also impurities from your viral supernatant can decrease the efficiency of infection We recommend using Cell Biolabs ViraBind Lentivirus Concentration and Purification Kit Catalog VPK 090 Measure the titer of your lentivirus This is an important step to ensure consistent viral transduction into your host cell However QPCR or stable clone counting can take as much as 1 2 weeks to perform Traditional p24 ELISA kits can greatly overestimate your lentiviral titer Our advanced p24 ELISA QuickTiter Lentivirus Titer Kit Catalog VPK 107 uses exclusive technology that eliminates free p24 from your supernatant givi
6. GCAGTGGCGCCCGAACAGGGACCTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCG CGCACGGCAAGAGGCGAGGGGCGGCGACTGCAGAGTACGCCAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCG GGGGAAAATAGCGGCCGCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTA AAGAATTACAAAAACAAATTACAAAAATTCAAATTTTCGGGGGATCCGCGGAATTCGTCGATATCAGCGTCGACAATCAACCTCTGGATTACAAAATTTGTGA AAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCC GTATCATGCTATTGCTTCCCGTATGGC CATT TTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCC CCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCC CCGGGAC CGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCG CTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATT CTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCC TTCGCCCTCAGACGAGTCGGATCTCCC GGGCCGCCTCCCCGCTTAGTACTGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACT TTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATTCCGGAATTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAA ACCGGTGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAA TAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTA GCATCTAGAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAA TTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGT
7. TCCGCCCATTCTCCGCCCCATGGCTGACTAATTTT TATT TATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTAGAGATCATAATCAGCCATACCACATT TGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTAT AATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATC ATGTCTGCTAGCCGGGC TTTTTCTTAGGCCTTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCAC TCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGT TGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGC GTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGC CTCAT AGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTA ACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGA
8. l Lentiviral Expression Vector Promoterless CELL BIOLABS INC A Unique Elements of the ViraSafe Lentivirus Expression System Third Generation Lentivirus Expression System ViraSafe Lentivirus Expression System TRANSFER VECTOR aus SD Femme I eer ce wes ene T Bid s PACKAGING VECTOR 1 PACKAGING VECTOR 2 ENVELOPE VECTOR i Vector Element Name Benefits compared to other 3 Generation Systems Name ELEMENTS ADDED Central e Increased gene expression levels Polypurine Tract Hybrid 3 LTR e Increased safety prevents read through transcription Transfer peel Poly A e Increased viral titer vector transcript more stable in packaging Vector cells were WPRE e Increased viral titer p Codon Wobble e Increased safety reduces sequence homology Packaging Vector 1 va Adenovirus VA e Increased viral titer ELEMENTS REMOVED Gag sequence e Increased safety reduces sequence homology Transfer ages Vector RRE Rev Responsive e Increased safety reduces sequence homology Element Kit Components 1 pSMPUW Universal Lentiviral Expression Vector Part No VPK 211 One 40 uL vial at 0 25 mg mL The plasmid is kanamycin resistant Note Please see Figure 2 for important instructions on bacterial culture of this plasmid pRSV Rev Packaging Vector Part No 320022 One 40 uL vial at 0 25 mg mL pCMV Eco E
9. 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 2009 2015 Cell Biolabs Inc All rights reserved No part of these works may be reproduced in any form without permissions in writing e CELL BIOLABS INC P A
10. ng you much more accurate lentiviral titers Results are obtained in 6 18 hours Use transduction reagents to increase infection efficiency Many cells are difficult to infect with lentivirus and without supplemental reagents transduction efficiencies can be low Reagents such as Polybrene can help but are often insufficient Cell Biolabs proprietary reagents in our ViraDuctin Lentivirus Transduction Kit Catalog LTV 200 form a super complex with your virus to increase transduction efficiencies by promoting virus and cell interaction CELL BIOLABS INC e Appendix pSMPUW Plasmid Sequence Pink 5 CMV LTR y cPPT Purple MCS Brown WPRE Orange 3 LTR Blue Kanamycin Resistance gene ACTAGTCGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATT GACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACAT CAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTAC GGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAG TCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGT AGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGGGTCTCTCTGGTTAGACCAGATTTGAGCCTGGGAGCTCTCTGGCTAACTAGGG AACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTT AGTCAGTGTGGAAAATCTCTA
11. nvelope Vector Part No 320026 One 40 uL vial at 0 25 mg mL pCgpV Packaging Vector Part No 320024 One 40 uL vial at 0 25 mg mL DE odes ocp wd pSMPUW LacZ Control Vector Part No 320025 One 40 uL vial at 0 25 mg mL containing a nuclear localized LacZ driven by MND retroviral LTR promoter The plasmid is kanamycin resistant Note Please see Figure 2 for important instructions on bacterial culture of this plasmid Materials Not Supplied 1 293T cells we recommend 293LTV Cell Line Cat LTV 100 for high titer production of lentivirus 2 Cell Culture Medium 3 Transfection Reagents Storage Upon receipt store all other 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 The ViraSafeTM Universal Lentiviral Expression System is designed to minimize the chance of generating replication competent lentivirus but precautions should still be taken to avoid direct contact with viral supernatants Preparation of p5MPUW Expression Vector pSMPUW Universal Expression Vector does not contain a promoter ahead of the multiple cloning sites nor does it contain any reporter genes or antibiotic selection markers You must clone a promoter into the vector along with your gene of interest An antibiotic selection marker and or reporter gene may
12. odate inserts up to 10 kb Key Features of ViraSafe Lentiviral Expression System 1 Transfer Plasmid Reduce extent of HIV sequences to increase capability up to 10 kb and reduce likelihood of recombination between vector components Add elements to increase titer and further improve safety 2 Packaging Plasmid Improve the packaging plasmid to increase performance and reduce the likelihood of recombination between vector components a Minimize HIV sequences no accessory proteins Tat or Rev or LTRs b Prevent overlap with vector SM by codon wobbling Gag sequences c Boost particle production by incorporating adenovirus VA element 3 Hlexible AII vectors including packaging vectors are provided separately to allow end user to optimize the vector ratio for maximal lentivirus production 2 CELL BIOLABS INC Packaging Vectors Envelope Transfer Vector Vector NTS Transfect into 293T Cells Harvest Viral Supernatant m Incubate with Target m Cells Lu md gt em 7 Figure 1 Lentivirus Production in 293T Cells Related Products LTV 100 293LTV Cell Line LTV 200 ViraDuctin Lentivirus Transduction Kit VPK 104 ViraBind Lentivirus Purification Kit VPK 107 QuickTiter Lentivirus Titer Kit Lentivirus Associated HIV p24 VPK 108 H QuickTiter Lentivirus Quantitation Kit HIV p24 ELISA VPK 205 ViraSafe Lentivirus Packaging System Ecotropic aye Se UB Pre VPK 211 pSMPUW Universa
13. tion competent lentivirus upon recombination In addition most commercial lentiviral packaging systems provide plasmids containing the viral structure proteins in a premixed formulation making it nearly impossible to optimize the ratio of the various plasmids for your particular experiment and host cell Also most commercial lentivirus transfer vectors contain promoters antibiotic selection markers and or reporter genes which may not be optimal or even suitable for your particular expression studies Cell Biolabs ViraSafe Universal Lentiviral Expression System provides a much safer method to package lentivirus while still providing high viral titers The sequence homology with native HIV 1 has been reduced by 80 90 even compared with other commercial third generation packaging systems In addition each plasmid is provided separately rather than in a packaging mixture This allows you the flexibility to amplify individual plasmids and optimize the ratio of plasmids for your experiment The expression vector in the ViraSafe Universal Lentiviral Expression System does not contain any promoter ahead of the multiple cloning sites nor does it contain any reporter genes or antibiotic selection markers This makes the system truly universal by allowing you to introduce your own promoter marker or reporter that is optimal for your gene of interest or target cell It also makes the system ideal for promoter studies The expression vector can accomm
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