Lentivector Expression Systems:
Contents
1. RAW 264 7 P19 mouse leukaemic monocyte macrophage mouse embryo teratocarcinoma 100 eee 100 9 FIV based pSIF1 copGFP 80 80 ti HIV based pSIH1 copGFP 2 E g 60 g 60 E E Q 40 2 40 t E z 20 FIV based pSIF1 copGFP 3 209 E HIV based pSIH1 copGFP 096 0 HHHH 0 1 2 3 4 5 6 7 8 9 410 012 3 4 5 6 7 8 9 10 11 12 13 14 15 Viral Titer arbitrary units Viral Titer arbitrary units NB41 NIH3T3 mouse neuroblastoma mouse embryonic fibroblast 100 80 2 2 g 8 60 E E 8 3 40 E FIV based pSIF1 copGFP a 20 FIV based pSIF1 copGFP HIV based pSIH1 copGFP HIV based pSIH1 copGFP 0 012 3 4 5 6 7 8 9 10 11 12 13 14 15 16 o 17 2 3 4 5 6 7 8 9 10 Viral Titer arbitrary units Viral Titer arbitrary units P388 mouse Lin ckit bone marrow mouse lymphocytic leukemia stem cells 100 9 FIV based pSIF1 copGFP 80 FIV based pSIF1 copGFP 80 88 HIV based pSIH1 copGFP E l HIV based pSIH1 copGFP E g 60 60 o o 2 2 Q 40 g 40 2 E E a 20 a 20 0 0 FH HH 0 1 2 3 4 5 6 7 8 9 10 0 5 10 15 20 25 30 35 40 45 50 55 Viral Titer arbitrary units Viral Titer arbitrary units 888 266 5066 Toll Free 650 968 2200 outside2. Day 3 4 Remove the culture medium and replace with 1 ml of complete medium without Polybrene Incubate the cells at 37 C with 5 CO overnight Day 4 5 By day 4 the culture will be confluent depending on cell type Split it 1 3 to 1 5 depending on the type of cells and continue incubating for 48 hours in complete D MEM Day 6 6 The infected target cells can be analyzed for transient expression of the expression construct using an appropriate biological assay If you have used an internal copGFP control determine the percentage of infected cells by counting fluorescing cells by flow cytometry Alternatively the infected cells can be identified by selecting and counting based on selection marker genes contained in the expression construct e g puromycin but the titer determined by puromycin selection is usually less than the titer determined by COpGFP selection Page 14 ver 080707 www systembio com Lentivector Expression Systems Cat s LV100A 1 LV601B 1 Guide to Packaging and Transduction of Target Cells amp LV900A 1 Ill Troubleshooting A Low Viral Titer lt 10 ifu ml 1 Poor Transfection Efficiency 293TN Cells have too high or too low density Plate fewer or more cells in order to have about 50 70 confluency at transfection stage Lentivector expression construct DNA preparation is of poor quality Purify plasmid DNA using a QIAGEN Endotoxin free Plasmid Purification Kit or by phenol chlorofo
3. which are provided with each of the Lentivector products can also be accessed on the SBI website http www systembio com Before using the reagents and material supplied with this product please read the entire user manual B Lentiviral Expression Systems Lentiviral expression vectors are the most effective vehicles for transducing and stably expressing different effector molecules siRNA cDNA DNA fragments antisense ribozymes etc or reporter constructs in almost any mammalian cell including non dividing cells and whole model organisms Cann 2000 As with standard plasmid vectors it is possible to introduce lentiviral constructs in plasmid form into the cells with low to medium efficiency and get transient expression of effectors reporters using conventional transfection protocols However by packaging the lentiviral expression construct into pseudoviral particles you can obtain highly efficient transduction up to 100 even with the most difficult to transfect cells such as primary stem and differentiated cells The expression construct transduced in cells is integrated into genomic DNA and provides stable long term expression of siRNA cDNA or reporter gene Endogenously expressed siRNA effectors provide long term silencing of the target gene and allow the researcher to generate cell lines and transgenic organisms with a stable knockdown phenotype for functional studies Expression of full length cDNAs from integrated viral
4. HIV based pSIH1 copGFP 2 2 g 60 8 o o 2 2 E 40 2 L 20 FIV based pSIF1 copGFP E 8 HIV based pSIH1 copGFP O Pe HHH 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 410 Viral Titer arbitrary units Viral Titer arbitrary units K562 HL60 human chronic myelogenous leukemia human acute myeloid leukemia 100 J A 100 80 80 FI V based pSIF1 copGFP o P l HIV based pSIH1 copGFP g 60 g 60 o o 2 2 Q 40 g 40 2 2 L 20 FIV based pSIF1 copGFP L 20 E HIV based pSIH1 copGFP 0 0 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 11 12 Viral Titer arbitrary units Viral Titer arbitrary units MOLT 4 THP 1 human acute lymphoblastic leukemia human acute monocytic leukemia 100 m 100 80 80 FIV based pSIF1 copGFP HIV based pSIH1 copGFP 2 2 g 60 8 60 o o 2 2 Q 40 Q 40 2 2 E X 20 FIV based pSIF1 copGFP X 20 E HIV based pSIH1 copGFP 0 0 4 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12 Viral Titer arbitrary units Viral Titer arbitrary units 888 266 5066 Toll Free 650 968 2200 outside US Page 23 System Biosciences SBI Human Primary Stem Cell Lines User Manual PBMC donor HUVEC 3
5. 11 12 2669 73 Brooks Al Stein CS Hughes SM Heth J McCray PM Jr Sauter SL Johnston JC Cory Slechta DA Federoff HJ Davidson BL Functional correction of established central nervous system deficits in an animal model of lysosomal storage disease with feline immunodeficiency virus based vectors Proc Natl Acad Sci U S A 2002 Apr 30 99 9 6216 21 Buchschacher G L and Wong Staal F 2000 Development of lentiviral vectors for gene theraphy for human diseases Blood 95 2499 2504 Burns J C Friedmann T Driever W Burrascano M and Yee J K 1993 Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors concentration to a very high titer and efficient gene transfer into mammalian and non mammalian cells Proc Natl Acad Sci USA 90 8033 8034 Curran MA Kaiser SM Achacoso PL Nolan GP Efficient transduction of nondividing cells by optimized feline immunodeficiency virus vectors Mol Ther 2000 Jan 1 1 31 8 Curran MA Ochoa MS Molano RD Pileggi A Inverardi L Kenyon NS Nolan GP Ricordi C Fenjves ES Efficient transduction of pancreatic islets by feline immunodeficiency virus vectors 1 Transplantation 2002 Aug 15 74 3 299 306 DePolo NJ Reed JD Sheridan PL Townsend K Sauter SL Jolly DJ Dubensky TW Jr VSV G pseudotyped lentiviral vector particles produced in human cells are inactivated by human serum Mol Ther 2000 Sep 2 3 218 22 888 266 5066 Toll Free 650 968 2200 outside
6. OTHER APPLICATION REQUIRES A LICENSE FROM EVROGEN To obtain such a license please contact Evrogen at license evrogen com SBI has pending patent applications on various features and components of the Product For information concerning licenses for commercial use contact SBI Purchase of the product does not grant any rights or license for use other than those explicitly listed in this Licensing and Warranty Statement Use of the Product for any use other than described expressly herein may be covered by patents or subject Page 28 ver 080707 www systembio com Lentivector Expression Systems Cat s LV100A 1 LV601B 1 Guide to Packaging and Transduction of Target Cells amp LV900A 1 to rights other than those mentioned SBI disclaims any and all responsibility for injury or damage which may be caused by the failure of the buyer or any other person to use the Product in accordance with the terms and conditions outlined herein Limited Warranty SBI warrants that the Product meets the specifications described in the accompanying Product Analysis Certificate If it is proven to the satisfaction of SBI that the Product fails to meet these specifications SBI will replace the Product or provide the purchaser with a refund This limited warranty shall not extend to anyone other than the original purchaser of the Product Notice of nonconforming products must be made to SBI within 30 days of receipt of the Product SBI s liability is expressl
7. US Page 17 System Biosciences SBI User Manual Derksen TA Sauter SL Davidson BL Feline immunodeficiency virus vectors Gene transfer to mouse retina following intravitreal injection J Gene Med 2002 Sep Oct 4 5 463 9 Dull T Zufferey R et al A third generation of lentivirus packaging system J Virol 1988 92 8468 8471 Gould D J and Favorov P 2003 Vectors for the treatment of autoimmune diseases Gene Therapy 10 912 927 Haskell RE Hughes SM Chiorini JA Alisky JM Davidson BL Viral mediated delivery of the late infantile neuronal ceroid lipofuscinosis gene TPP I to the mouse central nervous system Gene Ther 2003 Jan 10 1 34 42 Hughes SM Moussavi Harami F Sauter SL Davidson BL Viral mediated gene transfer to mouse primary neural progenitor cells Mol Ther 2002 Jan 5 1 16 24 Kang Y Stein CS Heth JA Sinn PL Penisten AK Staber PD Ratliff KL Shen H Barker CK Martins Sharkey CM Sanders DA McCray PB Jr Davidson BL In vivo gene transfer using a nonprimate lentiviral vector pseudotyped with Ross River Virus glycoproteins J Virol 2002 Sep 76 18 9378 88 Lotery AJ Derksen TA Russell SR Mullins RF Sauter S Affatigato LM Stone EM Davidson BL Gene transfer to the nonhuman primate retina with recombinant feline immunodeficiency virus vectors Hum Gene Ther 2002 Apr 10 13 6 689 96 Price MA Case SS Carbonaro DA Yu XJ Petersen D Sabo KM Curran MA Engel BC Margarian H Abko
8. US Page 25 System Biosciences SBI Rat Cell Lines User Manual C6 rat glioma cell line 100 r 80 60 40 infected cells 20 FIV based pSIF1 copGFP 8 HIV based pSIH1 copGFP 096 0 1 2 3 4 5 6 7 8 Viral Titer arbitrary units 100 80 infected cells D eo e e E E N o ES 0 RAT 1 rat embryonic fibroblast FIV based pSIF1 copGFP E HIV based pSIH1 c opGFP 2 3 4 5 6 7 Viral Titer arbitrary units Feline Cat Cell Line Hamster Cell Line Page 26 CHO Chinese hamster ovary CrFK cat domestic kidney cells 100 4 TT Il 100 80 80 9 60 8 60 o o 2 g Q 40 g 40 t 9 EFIV based pSIF1 copGFP t 9 FIV based pSIF1 copGFP we 20 i HIV based pSIH1 copGFP 39 20 HIV based pSIH1 copGFP 0 0 0 1 2 3 4 5 6 T7 8 9 10 0 1 2 3 4 5 6 T 8 9 10 Viral Titer arbitrary units Viral Titer arbitrary units Properties of the CopGFP Fluorescent Protein The pSIF1 H1 siLuc copGFP pSIH1 copGFP and pSIH1 H1 siLuc copGFP Vectors contain the full length copGFP gene with optimized human codons for high level of expression of the fluorescent protein from the CMV promoter in mammalian cells The copGFP marker is a
9. applications Poeschla 2003 Sodroski J G 1997 1999 Federico 2003 Heiser 2004 Machida 2003 Appendix A shows several examples of vectors for expression of siRNA pSI cDNA pCD and transcriptional reporters pTR These lentivectors have similar functional maps and include the following common features e Hybrid CMV 5 LTR for FIV based vectors and hybrid RSV 5 LTR promoter for HIV based vectors provide a high level of expression of full length pseudoviral constructs in 293 producer cells Genetic elements CPPT GAG LTRs necessary for the packaging transduction and stable integration of the viral expression construct into genomic DNA SV40 origin for stable propagation of the lentiviral plasmid in 293 producer cells The pUC origin for high copy replication and maintenance of the plasmid in E coli cells The ampicillin resistance gene for selection in E coli cells WPRE element enhances stability and translation of the CMV driven transcripts The SV40 polyadenylation signal enables efficient termination of transcription and processing of recombinant transcripts For siRNA Expression vectors e H1 expression cassette located in 3 ALTR provides constitutive and efficient RNA polymerase II dependent transcription of ShRNA transcript in a wide range of cell lines e CMV promoter promotes a high level of expression of copGFP fluorescent reporter puromycin N acetyl transferase drug selectable marker or truncated H2Kk prot
10. cell lines see Section E Relative titers may vary up to 10 to 20 fold depending on the chosen cell line Day 1 1 For each viral stock plate H1299 cells in a 24 well plate at a density of 0 6 1 x 10 cells per well 24 hours prior to viral infection Add 1 ml of complete D MEM medium with serum and antibiotics and incubate cells at 37 C with 5 CO overnight Day 2 2 Prepare complete D MEM medium plus 10 FBS with Polybrene to a final concentration of 5 ug ml Prepare an extra well as a control Note Polybrene is a polycation that neutralizes charge interactions to increase binding between the pseudoviral capsid and the cellular membrane The optimal concentration of Polybrene depends on cell type and may need to be empirically determined usually in the range of 2 10 ug ml Excessive exposure to Polybrene gt 12 hr can be toxic to some cells 3 Remove culture medium and replace with 0 5 ml of complete D MEM medium with 1096 serum and Polybrene from Step 2 For each pseudoviral stock use three wells Infect H1299 cells by adding 1 ul of viral stock into the first well dilution factor of 500 10 ul of viral stock into the second well dilution factor of 50 and 100 ul of viral stock into the third well dilution factor of 5 For one additional well mock well control add 0 5 ml of D MEM medium with Polybrene from Step 2 Incubate cells at 37 C with 5 CO overnight Day 3 4 Remove culture medium and repla
11. concentration of infection competent pseudoviral particles For these difficult to transduce cells it is probably desirable to select the clones stably expressing the Lentivector construct for experimental assays e SBI s Expression Lentivectors contain a deletion in the 3 LTR which leads to self inactivation of the lentiviral vector after reverse transcription and integration into genomic DNA Although more than one copy of a lentiviral construct may be integrated into the genome of a single cell the lentiviral construct cannot produce infectious viral particles However in spite of these safety features please remember that you are working with transducible pseudoviral particles Although the particles are replication incompetent they are infection competent so the expression cassette which they carry will infect integrate and express in any mammalian cells Please follow the recommended guidelines for working with BSL 2 safety class see Section I F for more details The following protocol provides a general recommendation for transduction of your lentivector expression construct packaged in pseudotyped viral particles into target cells Use these guidelines as a starting point for determining optimal conditions for your cells and experiments Day 1 1 Plate target cells in a 24 well plate at a density of 0 5 x 10 cells per well 24 hours prior to viral infection Add 0 5 ml of complete optimal medium with serum and antibiotics and
12. constructs is a unique tool to study gain of function effect for cellular phenotypes Stably integrated transcriptional reporter constructs are a novel approach to the study of transcriptional regulation in the natural chromosomal environment and the monitoring of specific signaling pathways Moreover lentiviral delivery does not produce the non specific cell responses typically associated with chemical transfections or use of an adenoviral delivery system Gould 2003 Cann 2000 SBI offers both FIV based and HIV based lentiviral expression systems Both systems consist of three main components 1 The lentiviral expression vector e g shRNA construct in pSIF1 H1 Puro or cDNA construct in pCDF lentivector 2 The lentiviral packaging plasmids e g pPACKH1 Packaging Plasmid mix 3 A pseudoviral particle producer cell line e g 293TN cells Page 2 ver 080707 www systembio com Lentivector Expression Systems Cat 4s LV100A 1 LV601B 1 Guide to Packaging and Transduction of Target Cells amp LV900A 1 The lentiviral expression vector contains the genetic elements required for packaging transduction stable integration of the viral expression construct into genomic DNA and expression of the siRNA cDNA or reporter The lentiviral packaging plasmids provide all of the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant pseudoviral particles For production of a high
13. incubate cells at 37 C with 5 CO2 overnight Note It is possible to use other plate formats for transduction as well In this case the amount of cells should be adjusted depending on the growth area of the well or plate Day 2 2 Prepare a mixture of complete medium with Polybrene at a final concentration of 5 ug ml Remove media from plate wells and replace with 0 5 ml of this Polybrene media mixture per well for 24 well plate Note Note Mix the virus with the medium gently by rotation or inversion Do not vortex Note Polybrene is a polycation that neutralizes charge interactions to increase binding between the pseudoviral capsid and the cellular membrane The optimal concentration of Polybrene depends on cell type and may need to be empirically determined usually in the range of 2 10 ug ml Excessive exposure to Polybrene gt 12 hr can be toxic to some cells 888 266 5066 Toll Free 650 968 2200 outside US Page 13 System Biosciences SBI User Manual 3 Infect target cells by adding the prepared virus We suggest using several amounts of pseudoviral stock example 1ul Sul and 10ul of virus In addition we recommend including a transduction well with a COpGFP positive control construct virus and other appropriate positive and negative control construct virus preparations Incubate cells at 37 C with 5 CO overnight For extremely fast growing and metabolizing cell lines such as 293T use 3 FBS in the medium
14. 000 and Buchschacher et al 2000 To ensure optimal results follow these general guidelines during your experiments e Lentiviral expression construct quality To generate your specific lentiviral expression construct refer to the protocol in the user manual provided with the vector Transfection efficiency significantly depends on the quality of plasmid DNA We recommend purifying plasmid DNA with a QIAGEN Endotoxin free Plasmid Kit see Section I F or by CsCl centrifugation You will need 2 ug of lentiviral expression construct in sterile TE buffer with a concentration ranging from 0 2 2 ug ul for each transfection in a 10 cm culture plate or 75 cm flask e Maintaining 293TN cell line The 293TN cell line is a highly transfectable derivative of the HEK293 cell line with constitutive expression of SV40 T antigen and neomycin resistance gene The 293TN cells should be grown at 37 C in a humidified chamber with 5 CO in D MEM medium supplemented with 4 mM L glutamine 1 5 g l glucose 100 units ml penicillin G 100 ug ml streptomycin 90 and heat inactivated fetal bovine serum 1096 With a doubling time of less than 24 hours the 293TN cells should be split every 1 2 days when they reach 70 80 confluency For subculturing detach the cells with 0 25 trypsin 0 03 EDTA at 37 C add fresh culture medium and split at a ratio of 1 3 1 5 e pSIF1 Hi siLuc CopGFP or pSIH1 H1 siLuc copGFP Expression Vector Included in t
15. CKH1 GAG 11 1 kb pPACKH 1 REV 5 5 kb AmpR Bla Promoter pVSV G 5880 bp VSV G ORI Lac Promoter CMV Promoter Fig 7 Functional maps of plasmids in pPACKH1 Packaging Plasmid Mix pPACKH1 GAG top left and pPACKH1 REV top right packaging plasmids and pVSV G pseudotyping plasmid bottom D Transduction Efficiencies of Different Cell Lines with Increasing Relative Concentration of Viral Particles for FIV based and HIV based Lentivectors Human Cell Lines 888 266 5066 Toll Free 650 968 2200 outside US Page 21 System Biosciences SBI User Manual H1299 UMUC 3 human non small cell lung carcinoma i009 human bladder carcinoma 100 o 80 80 o 2 8 60 60 mo 3 2 Q 40 Q 40 o 2 FIV based pSIF1 copGFP 20 4 8 HIV based pSIH1 copGFP a 20 Flv pased pel copoEle f8 HIV based pSIH1 copGFP 096 Het Een 096 012 3 4 5 6 7 8 9 10 11 12 13 14 15 0 1 2 3 4 5 6 7 8 9 10 11 Viral Titer arbitrary units Viral Titer arbitrary units 293 T BM HeLa S3 human embryonic kidney human c
16. HIV based shRNA Cloning and Expression Lentivector Included as a positive control in the pPACKH1 packaging kit to monitor packaging efficiency CMV s LTR AmpR RRE cPPT CMV pCDF1 MCS2 EFl copGFP promoter Cat CD111B 1 Mig 6 771 bp E MCS2 SV40 ORI promoter Fig 4 FIV based cDNA Expression Lentivector 888 266 5066 Toll Free 650 968 2200 outside US Page 19 System Biosciences SBI User Manual Nde l RSV S LTR i Multiple Amph Cloning Site for TRE RRE Y pTRH1 mCMV dscGFP i 72 m 6 801 bp 7 Nba UCORI 4 4 TDCOnDA 1 a pe P Cats TRSOOPA 1 AE cvy BamHI SV40 ORI SV40 Poly A 3 ALTR WPRE Fig 5 HIV based PathNet Transcriptional Reporter Lentivector B Functional Maps of Packaging Plasmids for FIV based Lentivector System CMV I E Bla Promoter gag gp1 x Amp pFIV 34N 10508 b pVSV G SV40Poly A nefap6 p 5880 bp Rev2 ORI RRE god VIF gp3 orf1 pol gp2 Lac Promoter R ees ev1 gp4 orf2 CMV Promoter Fig 6 Functional maps of plasmids in pPACKF1 Packaging Plasmid Mix pFIV 34N packaging plasmid left and pVSV G pseudotyping plasmid right Page 20 ver 080707 www systembio com Lentivector Expression Systems Cat 4s LV100A 1 LV601B 1 Guide to Packaging and Transduction of Target Cells amp LV900A 1 C Functional Maps of Packaging Plasmids for HIV based Lentivector System pUC ORI CMV promoter Amp h gag CMV promoter pUC ORI rev pPA
17. SSBI System Biosciences Lentivector Expression Systems Guide to Packaging and Transduction of Target Cells Cat s LV100A 1 LV201B 1 LV500 510A 1 LV600A 1 LV601B 1 LV900A 1 User Manual See PAC or kit label for storage temperature A limited use label license covers this product By use of this product you accept the terms and conditions outlined in the Licensing and Warranty Statement ver 080707 contained in this user manual Lentivector Expression Systems Cat 4s LV100A 1 LV601B 1 Guide to Packaging and Transduction of Target Cells amp LV900A 1 Contents l Introduction and Background A Purpose of this Manual sss 2 B Lentiviral Expression Systems 2 C SBI s Expression Lentivectors eese 3 D Packaging of Expression Constructs into Pseudoviral Particles 3 E Delivery of Packaged Lentivector Constructs into Target Cells 4 F Safety Guidelines sss 5 G ListofComponents sss 7 H Additional Required Materials sss 8 ll Protocol A Procedure Outline and General Commen 9 B Pseudovirus Production sss 10 C Pseudoviral Titer Estimation 11 D Transduction of the Packaged Lentivector Expression Construct 13 Ill Troubleshooting A Low Viral Titer lt 10 ifuml sss 15 B Inefficient Transduction of Packaged Constructs 15 IV References 17 V Appendix A Examples of SBI s Expression Lentivectors 19 B Functional Maps of Pack
18. aging Plasmids for FIV based System 20 C Functional Maps of Packaging Plasmids for HIV based System 21 D Percentage of Transduced Cells with Increasing MOI 21 E Properties of copGFP Fluorescent Protein 26 F Related Products sss 27 G Technical Support suu 27 VI Licensing and Warranty Statement a 28 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI User Manual I Introduction and Background A Purpose of this Manual This manual provides information describing how to package lentivector expression constructs in pseudoviral particles and use of packaged expression constructs for transduction of target cells Specifically it provides critical instructions on how to package an FIV based or HIV based Lentivector Expression construct in VSV G pseudotyped viral particles by co transfecting 293TN Producer Cells with a Lentivector Expression construct and the pPACKF1 for FIV based constructs or pPACKH1 for HIV based constructs Packaging Plasmid Mix Recommendations are also provided for selection and use of FIV based and HIV based lentivector systems for transducing a wide range of target cells This manual does not include information about construction of expression constructs in lentiviral expression vectors Information on making constructs using these vectors is available in the user manuals for each of SBI s Lentivector Cloning and Expression Vectors User manuals
19. anual VI Licensing and Warranty Statement Limited Use License Use of the pPACK Lentivector Packaging Kit Packaged Positive Transduction Control or 293TN Producer Cell Line i e the Product is subject to the following terms and conditions If the terms and conditions are not acceptable return all components of the Product to System Biosciences SBI within 7 calendar days Purchase and use of any part of the Product constitutes acceptance of the above terms The purchaser of the Product is granted a limited license to use the Product under the following terms and conditions The Product shall be used by the purchaser for internal research purposes only The Product is expressly not designed intended or warranted for use in humans or for therapeutic or diagnostic use The Product may not be resold modified for resale or used to manufacture commercial products without prior written consent of SBI This Product should be used in accordance with the NIH guidelines developed for recombinant DNA and genetic research FIV Vector System This Product is for non clinical research use only Use of this Product to produce products for sale or for any diagnostic therapeutic clinical including pre clinical veterinary or high throughput drug discovery purpose the screening of more than 10 000 compounds per day is prohibited In order to obtain a license to use this product for these commercial purposes contact The Regents of the Univers
20. ce with 1 ml of complete D MEM medium without Polybrene Incubate the cells at 37 C with 5 CO overnight Day 4 5 Split the cells 1 3 to 1 5 if necessary depending on the type of cells and incubate in complete D MEM for an additional 24 hours 888 266 5066 Toll Free 650 968 2200 outside US Page 11 System Biosciences SBI User Manual Day 5 6 Page 12 Count the fraction of fluorescent cells by FACS analysis You may also visualize the cells for copGFP fluorescence but the results may be inaccurate due to inconsistencies in counting methods Use an average of the fraction of green glowing cells in 5 10 random fields of view to estimate the overall fraction of fluorescing cells on the plate e the fraction of infected cells Multiply the fraction of infected cells by 1 5 x 10 in this example the expected number of H1299 cells on the plate at the moment of infection and by the corresponding dilution factor then divide into 0 5 ml to determine the relative titer of the pseudovirus in the supernatant Alternative Methods The viral titer can also be estimated by real time PCR using SBI s UltraRapid Lentiviral Titer Kit Cat LV960A 1 human cells or Cat LV960B 1 mouse cells If you are using an expression vector with puromycin resistance titer can be determined by puromycin selection Split the counted number of cells into aliquots in serial dilutions 1 10 1 107 1 10 1 10 and culture them in co
21. ein cell surface marker for detection and selection of transduced cells For cDNA Expression vectors e CMV promoter drives a high level of expression in a wide variety of cell lines of a gene of interest CDNA which may be cloned into the multiple cloning site MCS located downstream of CMV promoter e Optional second expression cassette with puromycin resistance gene or copGFP reporter under the control of a constitutive elongation factor 1 EF1 promoter for selection or FACS analysis of transduced cells For Transcriptional Reporter vectors e CMV promoter is replaced with a hybrid TRE mCMV promoter to drive expression of a reporter gene dscopGFP luciferase or B galactosidase depending on presence of specific transcriptional factor which binds to TRE element and activates transcription from the mCMV promoter For detailed descriptions of SBl s Expression Lentivectors please refer to the User Manual for each specific lentivector or visit SBl s web site at http www systembio com D Packaging of Expression Constructs into Pseudoviral Particles 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual Currently the most efficient technology for producing a high titer of infectious replication incompetent lentiviral particles is based on transient coordinated expression of a lentiviral expression construct and all necessary packaging proteins delivered into producer cells by simultaneous t
22. ervix carcinoma 100 100 LE TT LEI 80 80 2 2 g 60 g 60 oO oO 2 2 g 40 Q 40 t t E FIV E Ze 20 e FIV based pSIF1 copGFP 2 20 Pea o E HIV based pSIH1 copGFP z paseo pa Rit cop 096 4 096 HH H 012 3 4 5 6 7 8 9 10 11 12 13 14 15 0123 45 6 7 8 9 101112 13 14 15 16 17 Viral Titer arbitrary units Viral Titer arbitrary units HEPG2 BT 474 human hepatocellular liver carcinoma human breast ductal carcinoma 10096 100 Sick 8096 8096 o 2 g 60 B 60 o g E 40 Q 40 t t 39 20 9 FIV based pSIF1 copGFP Ss 20 FIV based pSIF1 copGFP E HIV based pSIH1 copGFP HIV based pSIH1 copGFP 0 0 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9g 410 Viral Titer arbitrary units Viral Titer arbitrary units Page 22 ver 080707 www systembio com Lentivector Expression Systems Guide to Packaging and Transduction of Target Cells Human Cell Lines cont d Cat s LV100A 1 LV601B 1 amp LV900A 1 MCF 7 OVCAR 3 human breast adenocarcinoma human ovarian adenocarcinoma 100 d FIV based pSIF1 copGFP 80
23. es VSV G protein pseudotyped mediate viral entry through lipid binding and plasma membrane fusion and can infect both mammalian and non mammalian cells Burns 1993 The pPACKH1 Packaging Plasmid Mix consists of an optimized mixture of three plasmids pPACKH1 GAG pPACKH1 REV and pVSV G see maps in Appendix C Similar to the FIV based Packaging Plasmid Mix the pPACKH 1 plasmids provide all necessary structural replication and integration proteins and helper functions to produce VSV G pseudotyped particles For more detailed descriptions and sequence information regarding our cloning and packaging lentivectors visit our web site at http www systembio com E Delivery of Packaged Lentivector Constructs into Target Cells Pantropic VSV G pseudotyped viral particles containing the RNA copy of the lentivector expression construct can be efficiently used to deliver and stably express effector and reporter sequences in a wide range of mammalian target cells In order to provide guidelines for the use of lentivector delivery systems we compared transduction efficiencies of FIV based and HIV based vectors in different cell types Fig 1 shows a comparison of transduction efficiencies of FIV based and HIV based lentivector systems for 27 different cell lines including primary and stem cells Page 4 ver 080707 www systembio com Lentivector Expression Systems Cat 4s LV100A 1 LV601B 1 Guide to Packaging and Transduction of Target Cells am
24. eschla EM FIV Vectors Methods Mol Biol 2003 229 251 71 Machida C A ed Viral vectors for gene therapy Methods and Protocols 2003 Humana Press Naldini L Lentiviruses as gene transfer agents for delivery to non dividing cells Curr Opin Biotechnol 1998 Oct 9 5 457 63 Sauter SL Gasmi M FIV vector systems Somat Cell Mol Genet 2001 Nov 26 1 6 99 129 Sauter SL Gasmi M Dubensky TW Jr A highly efficient gene delivery system derived from feline immunodeficiency virus FIV Methods Mol Med 2003 76 405 32 Prototypes of SBI s Lentivectors Poeschla EM Wong Staal F Looney DJ Efficient transduction of nondividing human cells by feline immunodeficiency virus lentiviral vectors Nat Med 1998 Mar 4 3 354 7 Poeschla E M Looney D J and Wong Staal F 2003 Lentiviral nucleic acids and uses thereof US Patent NO 6 555 107 B2 Sodroski J G Vector containing HIV packaging sequences packging defective HIV vectors and uses thereof US patent 5 665 577 1997 September 9 Sodroski J G Vectors containing HIV packaging sequences packaging defective HIV vectors and uses thereof US patent 5 981 276 1999 November 9 Delivery of Lentiviral Expression Constructs with Lentivector Systems into Target Cells Alisky JM Hughes SM Sauter SL Jolly D Dubensky TW Jr Staber PD Chiorini JA Davidson BL Transduction of murine cerebellar neurons with recombinant FIV and AAV5 vectors Neuroreport 2000 Aug 21
25. get cells We recommend using SBI s PEG it Virus Precipitation Solution Cat LV810A 1 Polybrene is toxic for target cells Optimize the concentration and exposure time to Polybrene during the transduction step It is also possible to transduce the cells without Polybrene though at a higher MOI No Expression of Expression Construct The CMV or H1 promoter is not functional in target cells We have observed this in primary cells but the only way to solve this problem is to change the type of target cells or replace the CMV promoter with the EF1 promoter and H1 promoter with the U6 promoter ver 080707 www systembio com Lentivector Expression Systems Cat s LV100A 1 LV601B 1 Guide to Packaging and Transduction of Target Cells amp LV900A 1 IV References FIV and HIV Lentivector System Reviews Cann A J ed 2000 RNA Viruses A Practical Approach Oxford Univ Press Curran MA Nolan GP Nonprimate lentiviral vectors Curr Top Microbiol Immunol 2002 261 75 105 Curran MA Nolan GP Recombinant feline immunodeficiency virus vectors Preparation and use Methods Mol Med 2002 69 335 50 Federico M Methods in Molecular Biology Volume 229 Lentivirus gene engineering protocols 2003 Humana Press Heiser W C ed Methods in Molecular Biology Volume 246 Gene delivery to mammalian cells Volume 2 Viral Gene transfer techniques 2004 Humana Press Loewen N Barraza R Whitwam T Saenz DT Kemler I Po
26. he pPACK Packaging Kit as a positive control to optimize and troubleshoot your packaging protocol Specifically this plasmid should be used as a reference when quantifying viral titer and assaying expression of your 888 266 5066 Toll Free 650 968 2200 outside US Page 9 System Biosciences SBI User Manual construct If you use a Lentivector without a copGFP reporter and expression of copGFP will not interfere with your biological assay you can mix the copGFP construct with your expression construct at a 1 100 ratio and use it as internal positive control at every stage of your experiment The vectors also express an shRNA targeting Firefly Luciferase Alternatively you may determine titer by RT PCR using one of SBI s UltraRapid Lentiviral Titer Kits LV960A 1 for human cells LV960B 1 for mouse cells Transfection with Lipofectamine and Plus reagent The protocol provided in the experimental section is based on our experience with lentivectors and 293TN cells and is similar to the one provided for Lipofectamine Invitrogen To maximize transfection efficiency you may need to optimize transfection conditions For a detailed protocol optimization of transfection conditions and for scaling up pseudoviral production please refer to Invitrogen s Lipofectamine Reagent Manual Pseudoviral titer The relative titer is measured based on flow cytometry or by fluorescent microscopy and is always specific to a particular cell l
27. he positive control copGFP construct or your packaged expression construct mixed with the copGFP construct for details see Section A To determine the concentration of pseudoviral particles required to provide the desired multiplicity of infection MOI for your target cells you should do several transductions with different concentrations of packaged pseudoviral particles containing the control plasmid Results of these test transductions should be used to determine an optimal concentration that yields the optimal percentage of infected cells based on copGFP fluorescence Note that some cell types e g some suspension cultures may be rather resistant to infection see Section I E e Expression of the lentiviral construct can be measured directly at about 48 72 hours after transduction transient transduction Selecting stably transduced cells requires additional time after transduction For example puromycin selection usually requires an additional two weeks The decision to use transiently transduced cells or selected cells depends on the nature of your target cells biological assay etc Some infected actively dividing cells e g 293 HT1080 HeLa etc may express the construct in 10096 of cells at an MOI of 1 For these easy to transduce cells most biological assays can be performed at 48 72 hours after transduction However some cells may only express the construct in 10 5096 of cells even when transduced with a high
28. ine For titering we use H1299 cells Below are some key terms used in the protocol ifu ml infectious units ml the relative concentration of infection competent pseudoviral particles MOI multiplicity of infection the ratio of infectious pseudoviral particles ifu to the number of cells being infected For example if 1 x 10 cells are to be infected at an MOI of 0 1 then 1 x 10 ifu should be added to the cells Transduction Efficiency the average copy number of expression constructs per genome of target cell in the infected transduced population B Pseudovirus Production The following protocol describes the generation of pseudoviral particles containing a copy of your lentivector expression construct The yield of recombinant lentiviral particles typically produced under these optimized conditions is 10 ml of 1 3 x 10 ifu ml for the FIV system and 2 5 x 10 ifu ml for the HIV system per 10 cm culture plate or 75 cm flask when measured by transduction of H1299 cells This amount of pseudoviral particles is generally sufficient to infect 5 10 x 10 target cells at an MOI equal to 1 We recommend including the copGFP expression construct as an external or internal positive control in all of your experiments 1 Page 10 Plate 3 x 10 293TN cells in a fresh 10 cm plate or 75 cm flask the day before transfection in 9 ml of D MEM medium supplemented with glutamine and heat inactivated serum 10 FBS witho
29. ion Control HIV based Cat LV601B 1 100 500 ul pSIH1 H1 siLuc copGFP Packaged Control 22 x 10 ifu may vary by lot e The Packaged Controls are shipped on dry ice and should be immediately stored at 70 C upon receipt Avoid thawing and refreezing of pseudoviral particles Each freeze thaw cycle causes reduction of the titer by 20 30 Properly stored pseudoviral particles are stable for 6 months from the date received 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SBI User Manual 293TN Producer Cell Line Cat LV900A 1 The 293TN Human Kidney cell line is optimized for effective production of a high titer of pseudoviral particles and stably expresses the SV40 large T antigen and neomycin gene products 0 5 2 ml 293TN Producer Cell Line 22 x 10 cells may vary by lot The 293TN Cell Line is shipped on dry ice and should be stored at 80 C or liquid nitrogen gas phase upon receipt Properly stored 293TN cells are stable for 1 12 months from the date received depending on storage conditions H Additional Required Materials SBI products 293TN Human Kidney Producer Cell Line SBI Cat LV900A 1 or ATCC 293T 17 Cat CRL 11268 Peg It Virus concentration solution SBI Cat LV810A 1 Lentivector Expression Construct see http www systembio com for a full list Expression constructs should be purified with a QIAGEN Endotoxin free Plasmid Purification Kit The following kit c
30. ity of California This Product or the use of this Product is covered by U S Patent No 6 555 107 owned by The Regents of the University of California HIV Vector System This product is for non clinical research use only Use of this Product to produce products for resale or for any diagnostic therapeutic clinical veterinary or food purpose is prohibited In order to obtain a license to use this Product for these commercial purposes contact the Office of Research and Technology Ventures at the Dana Farber Cancer Institute Inc in Boston Massachusetts USA This Product or the use of this Product is covered by U S Patents Nos 5 665 577 and 5 981 276 and foreign equivalents owned by the Dana Farber Cancer Institute Inc WPRE Technology SBI has a license to sell the Product containing WPRE under the terms described below Any use of the WPRE outside of SBI s Product or the Products intended use requires a license as detailed below Before using the Product containing WPRE please read the following license agreement If you do not agree to be bound by its terms contact SBI within 10 days for authorization to return the unused Product containing WPRE and to receive a full credit The WPRE technology is covered by patents issued to The Salk Institute for Biological Studies SBI grants you a non exclusive license to use the enclosed Product containing WPRE in its entirety for its intended use The Product containing WPRE is being transfe
31. kB dependent activation of a copGFP reporter gene 3 times more sensitive than competitor cell lines makes analysis of Nuclear Factor kappa B NF B pathway activation more sensitive and reliable e LentiMag Magnetotransduction Kit Cat LV800A 1 A novel simple and highly effective approach to increase transduction efficiency with SBI s lentiviral vectors compared to the standard method of Polybrene aided transduction e PEG it Virus Precipitation Solution Cat LV810A 1 Simple and highly effective means to concentrate lentiviral vector inocula produced with SBl s pPACK Lentivector packaging systems e Cloning and Expression Lentivectors many Choose from FIV and HIV based shRNA miRNA cDNA or TRE cloning and expression vectors For a list of currently available vectors please visit our website at www systembio com G Technical Support For more information about SBI products and to download manuals in PDF format please visit our web site http www systembio com For additional information or technical assistance please call or email us at System Biosciences SBI 1616 North Shoreline Blvd Mountain View CA 94043 Phone 650 968 2200 888 266 5066 Toll Free Fax 650 968 2277 E mail General Information info systembio com Technical Support tech systembio com Ordering Information orders systembio com 888 266 5066 Toll Free 650 968 2200 outside US Page 27 System Biosciences SBI User M
32. l microfuge tubes and store them at 80 C If you choose to concentrate the viral particles we recommend following the User Manual of the SBI s PEG it Virus Precipitation Solution Cat LV810A 1 Caution You are working with infectious pseudoviral particles at this stage Please follow the recommended guidelines for working with BSL 2 safety class see Section G for more details Note The supernatant containing the pseudoviral particles can be used directly to determine the pseudoviral titer and directly used to infect target cells in vitro as long as the target cells can survive in conditioned medium You can also aliquot and store the supernatant at 80 C without cryoprotectant ver 080707 www systembio com Lentivector Expression Systems Cat s LV100A 1 LV601B 1 Guide to Packaging and Transduction of Target Cells amp LV900A 1 Freezing and thawing may result in 20 30 loss of viral titer with each cycle Alternatively to obtain a higher titer you may concentrate pseudovirus by the any of the following suggested methods e SBI s PEG f Virus Precipitation Solution Cat LV810A 1 recommended e Centrifuge the supernatant at 50 000 x g for 90 min at 4 C then resuspend the pseudoviral containing precipitate in a smaller volume of D MEM e Sucrose cushion centrifugation C Pseudoviral Titer Estimation We recommend that you estimate the titer of the pseudovirus containing supernatant before proceedi
33. more effective at infecting several of the tested mouse cell lines P19 NB41 NIH3T3 P38 and some of the blood cells MOLT 4 K562 T cells from AML patient The HIV based system is more effective at infecting stem and primary cells HUVEC bone marrow adipose Pseudotyped lentiviruses have been successfully used to infect many other cell types including neuronal dendritic endothelial retinal pancreatic hepatic aortic smooth muscle cells airway epithelia skin fibroblasts macrophages etc Lentivectors have been successfully used also for direct in vivo delivery and expression of transgenes in muscle brain airway epithelium liver pancreas retina and skin For a more complete list of cells or tissues which have been successfully transduced with lentivectors please see the Reference Section The pseudoviral particles can infect or transduce target cells and express effector or reporter molecules but cannot replicate within target cells because the viral structural genes are absent and the LTRs are designed to be self inactivating upon transduction Following transduction into the target cells the expression cassette is reverse transcribed and integrated into the genome of the target cell After integration the expression cassette continuously and stably produces high levels of effector or reporter molecules in target cells Target cells stably expressing the effector molecule can be isolated using the selectable marker contained in
34. mplete D MEM medium containing puromycin 1 g ml Continue to incubate cells for one week Every second day replace the selection medium with fresh medium After at least a week determine the fraction of infected cells by calculating the number of colonies present at a given cell dilution multiplied by the cell dilution factor and divided into the number of cells taken for selection Then calculate relative viral titer as previously described Please note that the titer determined by puromycin selection is usually significantly less than the titer determined by counting GFP positive cells and it also depends on cell type and selection conditions For Lentivector constructs with the H2Kk reporter titer can be determined by staining transduced cells with FITC H2Kk antibodies followed by FACS analysis or fluorescent microscopy for details see http www miltenyibiotech com ver 080707 www systembio com Lentivector Expression Systems Cat s LV100A 1 LV601B 1 Guide to Packaging and Transduction of Target Cells amp LV900A 1 D Transduction of the Packaged Lentivector Expression Construct General considerations e The transduction efficiency of the expression construct varies significantly for different cells and experimental conditions including virus concentration exposure time to virus and growth area of cells In order to optimize transduction conditions we recommend that you use either a titered pseudoviral stock containing t
35. ng with transduction experiments for the following reasons e To ensure that pseudoviral stock is viable e To determine the percentage of target cells which can be transduced with pseudoviral stock e To control the number of copies of integrated viral constructs per target cell The protocol for measuring of relative titers assumes that the positive control expression plasmid the control copGFP plasmid was mixed with your expression construct as an internal control at a ratio of 1 100 and is packaged into pseudoviral particles In an alternative approach the copGFP control plasmid can be packaged separately but in parallel with your construct as an external control In this scenario the control plasmid would be used to check and optimize the transfection packaging steps see Section B If you are not using the copGFP plasmid as an internal control refer to the Alternative Methods note immediately following the titering procedure To determine the relative pseudoviral titer you will need to transduce the packaged lentivector expression construct into the H1299 cell line in the presence of Polybrene 4 8 ug ml for 3 8 hours allow 72 hours for expression to start and then count the number of cells expressing copGFP by fluorescent microscopy or by FACS To check your titer you can also choose a cell line appropriate for your experimental system The lentiviral constructs should effectively transduce most of the commonly used mammalian
36. novel natural green monomeric GFP like protein from copepod Pontellina sp The copGFP protein is a non toxic non aggregating protein with fast protein maturation high stability at a wide range of pH pH 4 12 that does not require any additional cofactors or substrates The copGFP protein has very bright fluorescence that exceeds at least 1 3 times the brightness of EGFP the widely used Aequorea victoria GFP mutant The copGFP protein emits green fluorescence with the following characteristics emission wavelength max 502 nm excitation wavelength max 482 nm quantum yield 0 6 extinction coefficient 70 000 M cm Due to its exceptional properties copGFP is an excellent fluorescent marker which can be used instead of EGFP for monitoring delivery of expression constructs into cells ver 080707 www systembio com Lentivector Expression Systems Cat 4s LV100A 1 LV601B 1 Guide to Packaging and Transduction of Target Cells amp LV900A 1 F Related Products e UltraRapid Lentiviral Titer Kit Cat LV960A 1 human cells LV960B 1 mouse cells Allows you to measure copy number MOI of integrated lentiviral constructs in genomic DNA of target cells after transduction with constructs made in any of SBI s FIV or HIV based Lentivectors or GeneNet siRNA Libraries e NF B 293 GFP Transcriptional Reporter Cell Line Cat TR800A 1 This human embryonic kidney HEK 293 based cell line with a 300 fold NF
37. ombinations can be used for Midi scale preparation of endotoxin free DNA gt QIAfilter Plasmid Midi Kit Cat 12243 and EndoFree Plasmid Maxi Kit Cat 12362 gt QIAfilter Plasmid Midi Kit Cat 12243 and EndoFree Plasmid Buffer Set Cat 19048 Please visit the QIAGEN website to download the specialized protocol that is not contained in the user manual gt http www1 giagen com literature protocols pdf QP15 pdf Additional reagents Page 8 Dulbecco s Modified Eagle s Medium D MEM high glucose with sodium pyruvate and L glutamine Invitrogen Cat 11995073 Fetal Bovine Serum Invitrogen Cat 16000036 Puromycin Sigma Cat P8833 Penicillin Streptomycin Invitrogen Cat 15070063 Trypsin EDTA Sigma Cat T3924 Polybrene hexadimethrine bromide Sigma Cat H9268 Millex HV 0 45 um PVDF filters Millipore Cat SLHVR25LS Tissue Culture Plates and Related Tissue Culture Supplies Lipofectamine Reagent Invitrogen Cat 18324 111 Plus Reagent Invitrogen Cat 11514 015 Sterile TE Buffer 10 mM Tris pH 8 0 0 1 mM EDTA pH 8 0 H1299 human lung carcinoma cell line ATCC Cat CRL 5803 ver 080707 www systembio com Lentivector Expression Systems Cat 4s LV100A 1 LV601B 1 Guide to Packaging and Transduction of Target Cells amp LV900A 1 ll Protocol A Procedure Outline and General Comments The flowchart below outlines the general steps required for packaging of b
38. oth FIV based and HIV based expression constructs and transduction and expression of the viral expression construct in target cells In the diagram below using the HIV based system as an example the positive control pSIH1 H1 siLuc copGFP plasmid which expresses the copGFP reporter is used as the expression construct To construct a lentiviral expression construct for your experiment refer to the user manual provided with each specific lentivector ETN Ff ON 4 Packaging pSIH1 H1 pVSV G et siLuc copGFP S N s a pPACK Packaging Plasmid Mix Expression x LO Construct Step 1 Co transfect 293TN cells with a lentiviral expression 293TN a he E 3 m construct and pPACK Producer K E i id mi es NC packaging plasmid mix s Step 2 Collect viral particles amp e determine the titer Pseudoviral 3 Lol Particles Xxx w Step 3 Infect target cells Step 4 Assay Cells e g by FACS analysis Fig 2 Procedure for transient production of pseudoviral particles and transduction of effector expression construct into target cells The lentiviral expression system was designed to simplify all necessary steps in production of pseudoviral particles and transduction of an expression construct into target cells For general information and background on working with lentiviral technology we recommend the General Reviews listed in the Reference Section particularly Federico 2003 Cann 2
39. p LV900A 1 Comparison of Transduction Efficiencies of FIV vs HIV in different cell lines at low MOI 12 8 6 9 E FIV based pSIF1 copGFP E HIV based pSIH1 copGFP ratio to H1299 o e Ss e N t e N o T M o eo co io x o o Q TITSE o amp OS gb ge 9 S8 gd 4 F 3t BSEet bt F Ss L os Gates SS Easa py 62725 8 8 BP E 608 SRRLEZESS IS 5 9 lil 50 Oo E Z a X208525 E 5 8 T 2 z z 28tcoct 5 Es o z TaLe p 9592992 E SER ESS human mouse rat cat S primary stem Fig 1 Comparison of transduction efficiencies for FIV based and HIV based packaged positive transduction controls for 27 different cell lines based on FACS analysis percentage of GFP positive cells at low Multiplicity of Infection MOI These data clearly demonstrate that unlike commonly used cancer cell lines like H1299 HeLa HeK295 HepG2 etc that can be effectively transduced by lentivectors some cell types mouse Lin ckit bone marrow P19 PBMC HL60 P388 are more resistant to infection More efficient transduction of more resistant cell types may be possible by using a higher concentration of pseudoviral particles per cell in order to achieve the same MOI as demonstrated in Appendix C but not in all cases It is important to mention that FIV based and HIV based lentivectors have different tropism For example the FIV based system is
40. pVSV G plasmids 0 5 ug ul 20 ul pSIF1 H1 siLuc copGFP Positive Control Plasmid 0 5 ug l pPACKH1 Lentivector Packaging Kit Cat LV500A 1 for packaging HIV based lentivector expression constructs 200 ul pPACKH1 Packaging Plasmid Mix Mixture of pPACKH1 GAG pPACKH1 REV and pVSV G plasmids 0 5 ug ul 20 ul pSIH1 H1 siLuc copGFP Positive Control Plasmid 0 5 ug ul e The pPACK Plasmid Mixes and copGFP Positive Control Plasmid are shipped on dry ice or blue ice and should be stored at 20 C upon receipt Properly stored plasmids are stable for 12 months from the date received Packaged Positive Transduction Controls Packaged VSV G pseudotyped Positive Transduction Controls are used to estimate and optimize transduction efficiency of lentivector expression constructs and packaged GeneNet siRNA Libraries The packaged positive controls with copGFP reporter are provided in an amount sufficient to infect 22 x 10 cells at an MOI of 1 The constructs contain an shRNA targeting Firefly Luciferase Proceed with Section II D for the transduction control protocol pSIF1 H1 siLuc copGFP Packaged Positive Transduction Control FIV based Cat LV201B 1 100 500 ul pSIF1 H1 siLuc copGFP Packaged Control 22 x 10 ifu may vary by lot pSIH1 copGFP Packaged Positive Transduction Control HIV based Cat LV600A 1 100 500 ul pSIH1 copGFP Packaged Control 22 x 10 ifu may vary by lot pSIH1 H1 siLuc copGFP Packaged Positive Transduct
41. passages donor peripheral blood mononuclear cells human umbilical vein endothelial cells 100 100 80 9 EFIV based pSIF1 copGFP 80 HIV based pSIH1 copGFP E 2 g 60 g 60 o o g g Q 40 Q 40 E E 8 20 3 20 9 FIV based pSIF1 copGFP il HIV based pSIH1 copGFP 0 0 HH i 0 1 2 3 4 5 6 7 8 9 10 012 3 4 5 6 7 8 9 10 11 12 13 14 15 Viral Titer arbitrary units Viral Titer arbitrary units bone marrow human mesenchymal AML donor stem cells donor acute myelogenous leukemia 100 100 80 80 a 2 g 60 g 60 E 8 Q 40 8 40 t FIV based pSIF1 copGFP FIV based pSIF1 copGFP 20 8 HIV based pSIH1 copGFP 20 HIV based pSIH1 copGFP 096 096 i 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 55 Viral Titer arbitrary units Viral Titer arbitrary units Page 24 100 adipose tissue human mesenchymal stem cells donor 80 60 40 infected cells 20 FV based pSIF1 copGFP HIV based pSIH1 copGFP 0 0 2 4 6 8 10 Viral Titer arbitrary units 12 14 16 18 20 22 24 ver 080707 www systembio com Lentivector Expression Systems Guide to Packaging and Transduction of Target Cells Cat s LV100A 1 LV601B 1 amp LV900A 1 Mouse Cell Lines
42. ransfection with lentiviral expression and packaging plasmids When expressed in packaging cells the highly efficient hybrid CMV 5 LTR or RSV S LTR promoter of the expression construct produces large numbers of the expression construct transcript that contains all of the functional elements e Psi RRE and cPPT required for efficient packaging The expression construct transcript is efficiently packaged into VSV G pseudotyped viral particles with helper proteins produced by the pPACK plasmids Pseudoviral particles generated by producer cells within 48 72 hr can be concentrated frozen and used in later experiments To facilitate packaging SBI offers a 293TN producer cell line that was optimized for effective production of a high titer of pseudoviral particles by introduction of the SV40 large T antigen and neomycin gene product The pPACKF1 Packaging Plasmid Mix consists of a combination of two plasmids pFIV 34N and pVSV G see maps in Appendix B The pFIV 34N plasmid contains the structural gag regulatory vif gp4 rev nef and replication pol genes which code for the proteins required to produce the lentivirus The viral env gene which encodes the envelope protein that defines the tropism e the range of infectable cells is deleted in the pFIV 34N construct The pVSV G plasmid expresses the envelope glycoprotein of vesicular stomatitis virus VSV G from the CMV promoter thus replacing lentiviral env gene Viral particl
43. rk with pseudoviral particles in a Class Il laminar flow hood e All procedures are performed carefully to minimize the creation of splashes or aerosols e Work surfaces are decontaminated at least once a day and after any spill of viable material e All cultures stocks and other regulated wastes are decontaminated before disposal by an approved decontamination method such as autoclaving Materials to be decontaminated outside of the immediate laboratory area are to be placed in a durable leakproof properly marked biohazard infectious waste container and sealed for transportation from the laboratory Page 6 ver 080707 www systembio com Lentivector Expression Systems Cat s LV100A 1 LV601B 1 Guide to Packaging and Transduction of Target Cells amp LV900A 1 G List of Components pPACK Lentivector Packaging Kits The pPACK Packaging Plasmid Mix is an optimized mixture of the packaging plasmids in an amount sufficient for 10 co transfections with a lentivector expression construct in 10 cm tissue culture plates or alternatively 75 cm flasks The positive control expression construct with copGFP reporter and shRNA sequence targeting Firefly luciferase is provided in an amount sufficient for 6 control co transfections with the pPACK packaging plasmid mix pPACKF1 Lentivector Packaging Kit Cat LV100A 1 for packaging FIV based lentivector expression constructs 200 ul pPACKF1 Packaging Plasmid Mix Mixture of pFIV 34N and
44. rm extraction followed by a CsCl gradient centrifugation Plasmid DNA Lipofectamine Plus Reagent ratios are incorrect Optimize the ratios using the guidelines provided in the Lipofectamine protocol Inefficient Production of the pseudovirus 293TN Cells are of poor quality Optimize growth conditions check growth medium and don t grow 293TN cells for more than 20 passages Check for mycoplasma contamination Do not overgrow the cells do not allow the cells to reach more than 90 confluency in order to keep the culture continuously in logarithmic growth phase Pseudoviral supernatant harvested too early or too late Harvest supernatant every 12 hours starting 24 hours after transfection for 2 3 days 24 36 48 60 72 hours then titer each batch Lentiviral expression construct is too large The packaging limit for the lentiviral system is 8 5 kb from 5 LTR to 3 dLTR However the efficiency of packaging drops significantly at greater than 2 kb of cDNA insert length For a 3 kb insert the titers could be 10 fold lower than for a 1 kb insert Truncated viral RNA transcript Re check the lentivector construct sequence to confirm the absence of a polyadenylation ATAAA site between the LTR elements B Inefficient Transduction of Packaged Lentivector Expression Constructs 1 Poor Infection Efficiency Your stock contains low titer of virus Optimize infection protocol by using standard pre packaged pseudoviral stock
45. rred to you in furtherance of and reliance on such license Any use of WPRE outside of SBI s Product or the Product s intended use requires a license from the Salk Institute for Biological Studies This license agreement is effective until terminated You may terminate it at any time by destroying all Products containing WPRE in your control It will also terminate automatically if you fail to comply with the terms and conditions of the license agreement You shall upon termination of the license agreement destroy all Products containing WPRE in you control and so notify SBI in writing This License shall be governed in its interpretation and enforcement by the laws of California Contact for WPRE Licensing The Salk Institute for Biological Studies 10010 North Torrey Pines Road La Jolla CA 92037 Attn Office for Technology Management Phone 858 435 4100 extension 1275 Fax 858 450 0509 CMV Promoter The CMV promoter is covered under U S Patents 5 168 062 and 5 385 839 and its use is permitted for research purposes only Any other use of the CMV promoter requires a license from the University of lowa Research Foundation 214 Technology Innovation Center lowa City IA 52242 CopGFP Control This product contains a proprietary nucleic acid coding for a proprietary fluorescent protein s intended to be used for research purposes only Any use of the proprietary nucleic acids other than for research use is strictly prohibited USE IN ANY
46. s of copGFP positive control which can be purchased from SBI see Appendix F Related Products Volume of infecting supernatant is too high Keep volume as low as possible to achieve maximal adsorption of viral particles to the cells The assay is performed too early Normally the maximal expression of integrated provirus is expected to develop by 72 hours after infection However some cells display delayed expression Try the assay at a later time such as 96 hours CMV promoter is not functional in target cells Replace the CMV promoter with the elongation factor 1 EF1 promoter in the expression construct Target cell line may be difficult to transduce Check titer with 293TN or H1299 cells Optimize the transduction protocol Use a higher MOI Concentration of Polybrene added during titration is too high Add and optimize Polybrene concentration in the range of 4 10 ug ml Loss of pseudoviral titer during storage Aliquot and store pseudoviral stock at 80 C Each freeze thaw cycle drops the titer about 30 Use a fresh aliquot for transduction 888 266 5066 Toll Free 650 968 2200 outside US Page 15 System Biosciences SBI User Manual Page 16 The cell might methylate some toxic sequences within 10 14 days Infection Affects Target Cell Viability Pseudoviral stock medium affects target cell growth Dilute the stock medium or concentrate the pseudovirus by centrifugation to minimize the amount of medium added to the tar
47. the expression vector construct e g puromycin or copGFP F Safety Guidelines SBI s Expression lentivectors together with the pPACK packaging plasmids comprise the third generation lentiviral expression system The HIV based lentivectors are based on the vectors developed for gene therapy applications by Dr J G Sodroski US patent 45 665 577 and 5 981 276 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual The original FIV expression system was developed by Eric M Poeschla David J Looney and Flossie Wong Staal at UCSD Poeschla 1998 Poeschla 2003 The feline immunodeficiency virus FIV was originally isolated from cat blood Despite common close exposure of humans to FIV through contact with domestic cats including bites scratches etc no human infection or disease has ever been associated with FIV Poeschla 2003 Both FIV based and HIV based lentivector systems are designed to maximize their biosafety features which include e A deletion in the enhancer of the U3 region of 3 ALTR ensures self inactivation of the lentiviral construct after transduction and integration into genomic DNA of the target cells e The CMV promoter in FIV based vectors and RSV promoter in HIV based vectors upstream of 5 LTR in the lentivector allow efficient Tat independent production of viral RNA reducing the number of genes from HIV 1 that are used in this system e Number of lenti
48. titer of pseudoviral particles producer cells e g HEK 293 cells need to be transiently co transfected with the expression and packaging vectors Expression constructs packaged in pseudoviral particles are secreted by producer cells in culture media and can be used directly to transduce expression constructs into target cells Following transduction into the target cells this expression construct is reverse transcribed and integrated into the genome of the target cell providing a high level of expression of effector or reporter molecules The most popular lentiviral expression system is HIV based Federico 2003 Heiser 2004 Machida 2003 Despite improved biosafety features third generation HIV cloning vectors still pose a potential biohazard risk due to the possible recombination with endogenous viral sequences to form a self replicating HIV virus SBI s FIV based Expression system addresses these issues since they are derived from a feline immunodeficiency virus Curran 2002 Sauter 2001 Loewen 2003 Both of SBI s HIV based and FIV based lentivector systems meet Biosafety Level 2 BSL 2 based on the criteria published by the Centers for Disease Control for details see section F C SBI s Expression Lentivectors SBI offers a wide range of FIV based and HIV based lentivectors for cloning and expression of siRNA cDNA and transcriptional reporters SBs lentivectors are a third generation of lentivectors developed for gene therapy
49. ut antibiotics Disperse the cells by thorough pipetting to ensure even distribution At the moment of transfection the cells should reach 50 70 confluency Mix 20 ul 10 ug of the pPACK Packaging Plasmid Mix with 2 ug 2 25 ul depending on concentration of your lentivector expression construct then dilute the plasmid mixture into 400 ul D MEM medium without serum and antibiotics Add 20 ul of Plus Reagent mix and incubate at room temperature for 15 min Dilute 30 ul of Lipofectamine Reagent into 400 ul of D MEM medium without serum and antibiotics Mix gently Dropwise add diluted Lipofectamine Reagent from step 3 to DNA Plus Reagent complex from step 2 then mix gently by inversion and incubate at room temperature for 15 min Add the DNA Plus Reagent Lipofectamine Reagent complex from step 4 to the plate from step 1 and mix complexes with medium gently by tilting the plates a few times and incubate at 37 C in a CO incubator for 48 hours We recommend collecting supernatants at 48 hours post transfection Step 5 Usually the peak of virus production is achieved at 48 hours Collect all 10 ml of the pseudovirus containing medium in a 15 ml sterile capped conical tube and centrifuge at 3000 rpm at room temperature for 5 minutes to pellet cell debris Following centrifugation transfer the viral supernatant into a fresh 15 ml sterile capped conical tube Then aliquot the supernatant into sterile 1 5 m
50. viral genes necessary for packaging replication and transduction is reduced to three gag pol rev and the corresponding proteins are expressed from different plasmids for HIV based packaging plasmids lacking packaging signals and share no significant homology to any of the expression lentivectors pVSV G expression vector or any other vector to prevent generation of recombinant replication competent virus e None of the HIV 1 genes gag pol rev will be present in the packaged viral genome as they are expressed from packaging plasmids lacking packaging signal therefore the lentiviral particles generated are replication incompetent e Pseucoviral particles will carry only a copy of your expression construct Despite the above safety features use of HIV based vectors falls within NIH Biosafety Level 2 criteria due to the potential biohazard risk of possible recombination with endogenous viral sequences to form self replicating virus or the possibility of insertional mutagenesis For a description of laboratory biosafety level criteria consult the Centers for Disease Control Office of Health and Safety Web site at http www cdc gov od ohs biosfty bmbl4 bmbl4s3 htm It is also important to check with the health and safety guidelines at your institution regarding the use of lentiviruses and always follow standard microbiological practices which include e Wear gloves and lab coat all the time when conducting the procedure e Always wo
51. witz JL Nolan GP Kohn DB Crooks GM Expression from second generation feline immunodeficiency virus vectors is impaired in human hematopoietic cells Mol Ther 2002 Nov 6 5 645 52 Sinn PL Hickey MA Staber PD Dylla DE Jeffers SA Davidson BL Sanders DA McCray PB Jr Lentivirus vectors pseudotyped with filoviral envelope glycoproteins transduce airway epithelia from the apical surface independently of folate receptor alpha J Virol 2003 May 77 10 5902 10 Stein CS Davidson BL Gene transfer to the brain using feline immunodeficiency virus based lentivirus vectors Methods Enzymol 2002 346 433 54 Wang G Sinn PL Zabner J McCray PB Jr Gene transfer to airway epithelia using feline immunodeficiency virus based lentivirus vectors Methods Enzymol 2002 346 500 14 Wang G Slepushkin V Zabner J Keshavjee S Johnston JC Sauter SL Jolly DJ Dubensky TW Jr Davidson BL McCray PB Jr Feline immunodeficiency virus vectors persistently transduce nondividing airway epithelia and correct the cystic fibrosis defect J Clin Invest 1999 Dec 104 11 R55 62 Page 18 ver 080707 www systembio com Lentivector Expression Systems Cat 4s LV100A 1 LV601B 1 Guide to Packaging and Transduction of Target Cells amp LV900A 1 V Appendix A Examples of SBI s Expression Lentivectors RSV 5 LTR AmpR RRE pSIH1 H1 copGFP ShRNA Vector CMV sv40 ORI Wen E SV40 Poly A Copt 3 ALTR y WPRE Fig 3
52. y limited to replacement of Product or a refund limited to the actual purchase price SBI s liability does not extend to any damages arising from use or improper use of the Product or losses associated with the use of additional materials or reagents This limited warranty is the sole and exclusive warranty SBI does not provide any other warranties of any kind expressed or implied including the merchantability or fitness of the Product for a particular purpose SBI is committed to providing our customers with high quality products If you should have any questions or concerns about any SBI products please contact us at 888 266 5066 2008 System Biosciences SBI 888 266 5066 Toll Free 650 968 2200 outside US Page 29
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