ViraSafe™ Lentiviral Bicistronic Expression
Contents
1. TACAAAAATTCAAATT I l GCCTTGGGGAATCCCAGGGACCGTCG CGTCATCACTGAGGTGGAGAAGAGCATGCGT l GGAGTTCCGCGT TGGCACCAAAAJ TGAGTGCTTCAAGI l TTCCAT l l ACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCAT l IGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACAT TGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACI TAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGI l CAATGGGAGTTTGTTT l CAACGGGACTI l GGTTTAGTGAACCGGGTCTCA Tm l ACGCCAAAAT TCGGGGGAT TGAGGCTCCGGI l GCCTAGAGAAGGTGGCGCGGGGTAAACT AAGTGCAGTAGTCGCCGTGAACGTTCGGCCGGCCAGATAT rC TAGTGI l ACAGT TGCCCGTCAGI TGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTI TGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCG l ITGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCG TGGGGGGI rCCGCCTCCCCGT AAACTCCCACT l GGGAAAGTGATGTCGTGTACA l CCCTTCGGACCAAGGGTCATI pr pr TACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAG l ICCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGT l CTGGTTAGACCAGATTTGAGCCTGGGAGCTCTCTGGCTAACTAGGG pr TGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTA TCACCACCCCCCCCAACCCGCCCCGACCGGAGCTGAGAGTAATTCAJ TAACGTAGAACCCAGAGATCGCTGCGTTCCCGCCCCCTCACCCGCCC TGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGG l GGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCG TAATTAAGAATTCCGCCCCTCTCCCTAACGTTACTG l AGGGGTCTTTCCCCTCT ACCCCATTGTATGGGATCT CATGACCCGCAAGCCCGGTGCCTGAGTCGACAATCAACCTCTGGA TAATGCCTTTGTATCATGCTATTGCTTCCCGTA2. Product Manual ViraSafe Lentiviral Bicistronic Expression System Puro Pantropic Catalog Number VPK 215 PAN 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 generati
3. BIOLABS INC Example of Results The following figure demonstrates typical results seen with Cell Biolabs ViraSafe Lentiviral Expression System One should use the data below for reference only Figure 6 GFP and nLacZ Lentivirus Production and Transduction Lentiviral supernatant is produced by cotransfecting 293T cells Cat LTV 100 with pLenti GFP Cat LTV 400 or pSMPUW MNDnLacZ Cat LTV 402 and ViraSafeTM Lentiviral Packaging System Cat VPK 206 293AD cells Cat AD 100 are seeded at 100 000 cells well in a 6 well plate overnight Cells were infected with GFP or nLacZ lentivirus in the presence of 8 ug mL Polybrene for 72 hrs Left 293LTV Transfection Right 293AD Transduction CELL BIOLABS INC P sme Appendix pSMPUW IRES Puro Plasmid Sequence Pink Blue EF 1 Purple MCS Red IRES Green Puro 5 CMV LTR w cPPT Brown WPRE Orange 3 LTR Blue Kanamycin Resistance gene ACTAGTCGGGGTCATTAGTTCATAGCCCATATAT GACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACT CAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGT CAATGACGGTAAAT GGCAGTACATCTACGTAT TCTCCACCCCATTGACGT AGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT AACCCACTGCTTAAGCCT AGTCAGTGTGGAAAATCT l CAATAAAGCTTGCCTT l CTAGCAGTGGCGCCCGAACAGGGACCT CGCACGGCAAGAGGCGAGGGGCGGCGACTGCAGAGT GGGGAAAATAGCGGCCGCCACAATTTTAAAAGAAAAGGGGGGATA AAGAATTACAAAAACAAAT ACAAAAGGACTCGCCCCA GCTC GGTCGGCAATTGAACCGGT TATA GCCGAAGCCGCTTGGAAT CTTCTTGACGAGCATTCCT
4. A l ITGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCC TATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATA TCGGGAAGCGT ATCATGTCTGCTAGCCGGGCTTTTTTTTCTTAGGCCTTCTTCCGCT TCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACG AAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCA TAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTI TCCGACC TGGCGCTTTCI 9 TCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCT CELL BIOLABS INC CCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCC ACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACA GTA GGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGG TTTG TTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGG GA GGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCC AAATTAAAAATGAAG AAATCAATCTAAAGTATATATGAGTAAACTTGGTCT GACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTA CGTTCATCCATAGTTGCCTGACTCCTGCGCAGTCCAAAAAAAAAGGCTC CAAAAGGAGCCTTTAATTGTATCGGTGGGCCCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTT GAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAAT ACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCT TTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAA
5. ACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATA CGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTC TTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAAT TCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACA ATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGA CGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAA CATCAGAGATTTTGAGACACAACGTGGTTTAAACAAATAGTCAAAAGCCTCCGGCG 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 KahlC A Marsh J Fyffe J Sanders D A and K Cornetta 2004 J Virol 78 1421 30 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 resea
6. CAGTTCCTCTGGAAGCTTCTTGAAGA l IGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTA GCTGTCTCTTTATGAGG GGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGG TGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTG TECECTTECGGCECTCAATCO l CAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCC GCTTAGTACTGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCA ACGAAGACAAGATTCCGGAATTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAAACCGGTGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTA GACCAGATCTGAGCCI TGTGTGACTCTGGT CAGGTGTGGAAAGT l AACT l SGGAGCTCTCTGGCTAACTAGGGAACCCACI AGAGATCCCTCAGACCCI TCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCA CCCCTAACTCCGCCCAG CCGCCCATTC AGTAGTGAGGAGGCTTT CTGAACCI l GAAACATAAAA TGGAGGCCT CCGCCCCATGGCTGACT TAGGCTAGAGATCATAATCAGCCATACCACAT l ITTAGTCAGT TGAATGCAATTGTTGT TGTGGAAAAI TGCTTAAGCCI C TCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGT r CTAGCATCTAGAGTATGCAAAGCATGCATCTCAATTAGTCAGCAAC C CAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCG TAATTTTT GTTAACTTG AAGCA TCCTCGCT TTTTCAC l CACTGACT CAAAAAT CTGCCGC TACCGGA GCA CGC1 ACCT TCTAGTTG GCGCTCGGT GGTTTG TCGTTCGGCI CCAAACTCAT TGTCCGCCTTTCTCCC l CAATGTATC l GCGGCGAGCGGTATCAGCT CAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGT TCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTAJ TATTGCAGCI ATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAG
7. TGGCTI l CAGGCAACGTGGCGTGGTGTGCACTGTGT TCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTI TCATCGTCCTTTCCTTGGCTGCTCGCCTGTGI PTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTC TGTGGATACGCTGCTA AGTTGTGGCCCGTTGT GACTTI GTGTTGTCGGGGAAAJ CAGCGGACCTTCC A l AAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCA TCGCCAAAGGAATGCAAGGTC CAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGG CGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGT l GATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTG GTTTTCCTTTGAAAAACACGATGATAATATGGCCACAACCATGGTTACCGAGTACAAGCCCACGGTGCGCCTCGCCACCCGCGACGACGTCCCCAGGGCCGTA CGCACCCTCGCCGCCGCGTTCGCCGACTACCCCGCCACGCGCCACACCGTCGATCCGGACCGCCACATCGAGCGGGTCACCGAGCTGCAAGAACTCTTCCTCA CGCGCGTCGGGCTCGACATCGGCAAGGTGTGGGTCGCGGACGACGGCGCCGCGGTGGCGGTCTGGACCACGCCGGAGAGCGTCGAAGCGGGGGCGGTGTTCGC CGAGATCGGCCCGCGCATGGCCGAGTTGAGCGGTTCCCGGCTGGCCGCGCAGCAACAGATGGAAGGCCTCCTGGCGCCGCACCGGCCCAAGGAGCCCGCGTGG TTCCTGGCCACCGTCGGCGTCTCGCCCGACCACCAGGGCAAGGGTCTGGGCAGCGCCGTCGTGCTCCCCGGAGTGGAGGCGGCCGAGCGCGCCGGGGTGCCCG CCTTCCTGGAGACCTCCGCGCCCCGCAACCTCCCCTTCTACGAGCGGCTCGGCTTCACCGTCACCGCCGACGTCGAGGTGCCCGAAGGACCGCGCACCTGGTG ACAAAAT l TGCCACCI CCGCGTCT l IGTGAAAGAT l ICATTTTCTCCTCCTTGTATAAATCCTGG GCTGACGCAACCCCCAC reCCCGCTGCT TGGATTCTGCGCGGGACGTCCTTCTGCTACGI TCGCCTTCGCCCT l ITTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGT TGTTGAATGTCGTGAAGGAAG
8. er ce wes were aa 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 ll Codon Wobble e Increased safety reduces sequence homology Packaging Vector 1 KI 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 M moo bs pSMPUW IRES Puro Lentiviral Expression Vector Part No VPK 215 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 VSV G Envelope Vector Part No RV 110 One 40 uL vial at 0 25 mg mL pCgpV Packaging Vector Part No 320024 One 40 UL vial at 0 25 mg mL 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 p
9. mprove 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 Flexible All vectors including packaging vectors are provided separately to allow end user to optimize the vector ratio for maximal lentivirus production CELL BIOLABS INC Packaging Vectors e nvelope Transfer Vector Vector Transfect into 293T Cells Harvest Viral Supernatant EY m Incubate with Target P rc Cells LE a Figure 1 Lentivirus Production in 293T Cells Related Products LTV 100 293LTV Cell Line LTV 200 ViraDuctin Lentivirus Transduction Kit LTV 300 GFP Lentivirus Control 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 PS I4 ve Dm ome du qo VPK 211 pSMPUW Universal Lentiviral Expression Vector Promoterless 3 CELL BIOLABS INC Unique Elements of the ViraSafe Lentivirus Expression System Third Generation Lentivirus Expression System ViraSafe Lentivirus Expression System TRANSFER VECTOR us SD Fawsfus I e
10. ng replication 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 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 pSMPUW IRES Puro Lentiviral Expression Vector contains EF 1a promoter ahead of the multiple cloning sites followed by an IRES and puromycin resistant gene Figure 2 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 I
11. oteinase per mL of bacterial lysate before adding neutralization solution RSV Rev Figure 3 pRSV Rev Packaging Vector 4180 bp Ampicillin resistant EcoRI Digestion 300 bp 3880 bp NA Figure 4 pCMV VSV G Envelop Vector 6051 bp Ampicillin resistant EcoRI Digestion 787 bp 1668 bp 3596 bp Figure 5 pCgpV Packaging Vector 9118 bp Ampicillin resistant Pst I Digestion 927 bp 1424 bp 6767 bp e CELL BIOLABS INC P 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 pCMV VSV G 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 you
12. r gene The following steps should be considered prior to infection of your host cell 1 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 giving 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
13. rch 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 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 10 a CELL BIOLABS INC OIU 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 wri
14. romoter 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 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 ViraSafe 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 pSMPUW IRES Puro Vector 5 LTR Kan pSMPUW cPPT IRES Puro JR 6 2 kb EF 1 WPRE Puro IRES MCS MCS AACGTTCGGCCGGCCAGATATCTCCCTTCGGACCAAGGGTCATTAATTAAGAATTCCGCCCCTCT FseI ECORV AhdI PacI EcoRI Figure 2 pSMPUW IRES Puro Lentiviral Expression Vector 6237 bp Kanamycin resistant EcoRI and Xhol Digestion 1687 bp 4550 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 competent cells Invitrogen and treatment with alkaline proteinase Promega A1441 or Sigma P8038 for 4 5 min using 10 units of pr
15. ting 11 e CELL BIOLABS INC OIU
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