LENTI-Smart. For the generation of lentiviral particles USER MANUAL. FOR RESEARCH PURPOSES ONLY

Transcription

1 LENTI-Smart For the generation of lentiviral particles FOR RESEARCH PURPOSES ONLY USER MANUAL Catalog # ltsint-5, ltsint-10, ltsnil-5, ltsnil-10 Version # 16K25-MM INVIVOGEN USA INVIVOGEN EUROPE INVIVOGEN ASIA 3950 Sorrento Valley Blvd, Suite I00, San Diego, California 92121, USA Toll-Free: Fax: info@invivogen.com Z.I. Montaudran 5 rue Jean Rodier Toulouse, FRANCE Tel: Fax: info@invivogen.fr Unit 709A, Bio-Informatics Center 2 Science Park West Avenue, HKSTP, Shatin, HONG KONG Toll-free: Fax: info.hk@invivogen.com

2 TABLE OF CONTENTS Lentiviral Vectors Product Description Advantages of LENTI-Smart Lentiviral Vector Production Content and Storage Related Products Additional Materials Required Key Terms Used Experimental Outline Safety Consideration Methods - Growth and maintenance of HEK 293T cells Lentiviral cloning plasmid Positive control for lentiviral vector production Production of lentiviral vectors using HEK 293T cells DNAse Treatment of supernatant containing lentiviral vectors Concentration of lentiviral vectors Titration of lentiviral vectors p24 ELISA Selecting a cell line for titering FACS analysis titration method Antibiotic selection titration method qrt-pcr titration method General considerations for transduction Transduction procedure LENTIVIRAL VECTORS Lentiviral vectors derived from the human immunodeficiency virus (HIV-1) have become major tools for gene delivery in mammalian cells. The advantageous feature of lentiviral vectors is the ability to mediate efficient transduction, integration and long-term expression into dividing and non-dividing cells both in vitro and in vivo (Bloomer et al., 1997; Naldini et al., 1996). Currently, production of high titers of lentiviral vectors is a time consuming, multi-step procedure with low reproducibility. LENTI-Smart has been designed to solve these problems, by creating a lyophilizate of optimized packaging plasmids combined with a DNA transfection reagent. Upon rehydration, this lyophilizate serves as a carrier for your favorite lentiviral cloning plasmid. In some situations, integration of the transgene into the host cell's chromosomes poses safety concerns due to the potential risk of insertional mutagenesis (Verma & Somia, 1997). Non-integrating lentiviral (NIL) vectors combine the advantageous features of lentiviral vectors with episomal replication alleviating the risk of insertional mutagenesis. Lentiviral vectors can be rendered integration defective by mutations in the integrase coding sequence or altering the integration recognition sequences (att sites) in the viral LTR (Nightingale et al., 2006). NIL vectors can mediate transient transduction in vitro and in vivo (Yanez-Munoz et al., 2006; Apolonia et al., 2009). Depending on your requirements, LENTI-Smart kits are available for the production of either integrating or NIL vectors. LENTI-Smart NIL kits are available featuring the mutated integrase D64V (Leavitt et al,. 1996; Nightingale et al., 2006) or alternatively the mutated integrase D116N (Negri et al., 2007). Appendix - Troubleshooting guide Map and features of plv-ivsv-g Map and features of plv-help Map and features of plv-green Use restrictions References

3 rev tat RRE GFP PRODUCT DESCRIPTION LENTI-Smart is a ready-to-use product that allows for rapid and reliable production of high titers of second generation lentiviral particles in HEK 293T cells. LENTI-Smart combines a mix of optimized packaging plasmids precomplexed to a transfection reagent, LyoVec, selected for its high transfection efficiency and low cell toxicity. This lyophilized complex acts as a carrier for your favorite lentiviral cloning plasmid (not provided). LENTI-Smart allows for the production of integrating lentiviral vectors. Following transduction with integrating lentiviral vectors, the transgene is integrated into the target cell genome and results in stable transgene expression. The two packaging plasmids forming the LENTI-Smart lyophilizate provide the structural and replication proteins in trans that are required for the production of the lentiviral particles: plv-ivsv-g expresses a viral envelop gene plv-help contains the gag, pol, rev and tat genes. LENTI-Smart NIL allows for the production of non-integrating lentiviral (NIL) vectors. Transduction with NIL vectors allows for transient transgene expression in dividing cells. In quiescent cells, such as neurons, NIL vectors will result in stable expression of the non-integrated transgene. The two packaging plasmids forming the LENTI-Smart NIL lyophilizate are: plv-ivsv-g expresses a viral envelop gene plv-help-nil contains the gag, pol (mutated), rev and tat genes. This plasmid expresses a defective integrase (D64V) abolishing integration activity. ADVANTAGES OF LENTI-Smart - CONSISTENT HIGH TITERS 1-5 x 10 6 transducing units (TU) per ml (unconcentrated) are commonly achieved - FAST Lentiviral production time reduced and you no longer need to produce the packaging and pseudotyping plasmids nor do you need to plate your cells 24 hours prior to lentiviral vector production - SIMPLE Everything is ready for your experiment, just add your favorite lentiviral expression plasmid - VERSATILE Works with most commercially available lentiviral expression plasmid - REPRODUCIBLE Fluctuating parameters are kept to a minimum. LENTIVIRAL VECTOR PRODUCTION To produce lentiviral vectors, essential lentiviral (HIV-1) genes need to be transfected into HEK 293T cells. These cells are typically transfected with three plasmids (i) the lentiviral expression plasmid, such as plv-green, containing the psi (Y) packaging sequence and the transgene gene inserted between the lentiviral LTRs for target cell integration, (ii) a packaging plasmid (plv-help) encoding pol, gag, rev and tat viral genes and the rev-response element (RRE), and (iii) a pseudotyping plasmid (plv-ivsv-g) encoding the G protein of the Vesicular Stomatitis Virus (VSV-G) envelope gene. VSV-G is used in order to extensively broaden the target cell range (Burns et al., 1993). To optimize safety, the HIV accessory genes have been eliminated from the second generation of lentiviral vectors. Two days after transfection of HEK 293T cells, the cell supernatant contains active lentiviral vectors, which can be used to transduce your target cells with your gene of interest. Control Lentiviral Cloning Plasmid Target cell Each LENTI-Smart kit includes a control self-inactivating (SIN) lentiviral expression plasmid designed to optimize virus production and cell transduction. plv-green contains a green fluorescent protein (GFP) gene. VSV-G plv-ivsv-g Ψ Recombinant protein (GFP) plv-help gag host genome When combined with LENTI-Smart to generate lentiviral particles and transduced in mammalian cells, it expresses GFP that can be easily visualized by fluorescence microscopy. pol 5 LTR Ψ RRE prom plv-green HEK 293T 3 LTR Ψ VSV-G envelop protein pol reverse transcriptase/integrase gag capsid protein psi packaging sequence 2 3

4 CONTENTS LENTI-Smart kits are available for the production of either integrating (INT) or non-integrating lentiviral (NIL) vectors. Each kit is provided in 2 different pack sizes: LENTI-Smart INT Kit for the generation of integrating lentiviral vectors - ltsint-5: 5 vials of LENTI-Smart INT and 1 vial of plv-green - ltsint-10: 10 vials of LENTI-Smart INT and 1 vial of plv-green LENTI-Smart NIL Kit for the generation of non-integrating lentiviral vectors - ltsnil-5: 5 vials of LENTI-Smart NIL and 1 vial of plv-green - ltsnil-10: 10 vials of LENTI-Smart NIL and 1 vial of plv-green Each vial of LENTI-Smart allows for the transfection of HEK 293T cells with a lentiviral expression plasmid in a 10-cm plate or a T-75cm 2 flask. All LENTI-Smart kits are provided with 1 vial containing 15 μg of the control lentiviral expression plasmid plv-green. STORAGE Products are provided lyophilized and shipped at room temperature. Upon receipt, store products at -20 C. Lyophilized LENTI-Smart and plv-green are stable for 1 year when stored at -20 C. Resuspended plv-green is stable for 1 year when stored at -20 C. Avoid repeated freeze-thaw cycles. RELATED PRODUCTS Product Description Quantity Cat. Code PlasmoTest Mycoplasma testing kit 1 kit rep-pt Plasmocin Mycoplasma treatment 50 mg ant-mpt Normocin Mycoplasma, bacterial and fungal prevention 500 mg ant-nr-1 Blasticidin Selective antibiotic 100 mg ant-bl-1 Zeocin Selective antibiotic 1 g ant-zn-1 Puromycin Selective antibiotic 100 mg ant-pr-1 ADDITIONAL MATERIALS REQUIRED Critical parameters for lentiviral production - Lentiviral cloning plasmid preferably high purity, self-inactiving (SIN) from a commercial supplier. - HEK 293T cells. Use healthy cells at low passage numbers and actively replicating. We recommend 293FT cells (Invitrogen, cat. code R700-07). Alternatively, 293 cells expressing the SV40 large T antigen such Lenti-X 293T (Clontech, cat. code ) or HEK 293T/17 (ATCC, cat. code CRL-11268), can be used for lentiviral production. - Absence of mycoplasma contamination. Test for mycoplasma using PlasmoTest. Mycoplasma contamination can be treated using Plasmocin. Normocin can be used to prevent mycoplasma, bacterial and fungal contamination. - Tissue culture plastics which enhance cell adherence such as Corning CellBIND, cat. code 3290 for a T-75 cm 2 flask. - Fetal bovine serum (FBS), the FBS used can affect lentiviral production. We recommend Thermo Scientific HyClone, cat. code SH Low binding protein filter, 0.45 mm Millex -HV (Millipore, cat. code SLHV033RK). The filter should be made of PVDF (low protein binding), instead of nitrocellulose. Nitrocellulose binds proteins present in the membrane of the lentivirus causing the virus to remain attached to the membrane following filtration. Also required for culture of HEK 293T cells are DMEM containing 4.5 g/l glucose, L-glutamine, sodium pyruvate, penicillin & streptomycin, non-essential amino acids, phosphate buffered saline (PBS), and Trypan Blue (0.4 % solution). Optional: For treatment of lentiviral containing supernatant: DNase I (RNase-free) and magnesium chloride (1M; molecular biology grade). For simple concentration of lentiviral vectors: Amicon Ultra 100 kda filter (Millipore, cat. code UFC910008). For titration: HT1080 Cells (ATCC, cat. code CCL-121) and Polybrene. For transduction: Polybrene or alternatively protamine sulfate in cells sensitive to the cytotoxic effects of Polybrene. KEY TERMS USED: - TU/ml (transduction units/ml): the relative concentration of transduction-competent lentiviral vectors. - MOI (multiplicity of infection): ratio of transduction-competent lentiviral vectors (TU) to the number of cells being transduced, e.g. if 1 x 10 6 cells are to be transduced at a MOI of 0.1, than 1 x 10 5 TU should be added to the cells. - Transduction efficiency: the average percentage of cells expressing the transgene after lentiviral transduction. - Transgene: gene of interest (GOI) with an internal promoter. - LTR: long terminal repeat, sequences implicated in transgene integration. - SIN lentiviral vector: a deletion in the 3 LTR leads to inactivation of the lentiviral vector after reverse transcription and integration into genomic DNA. 4 5

5 EXPERIMENTAL OUTLINE Lentiviral cloning plasmid Invivo LENT LENTI-Smart 1. Clone your gene of interest into your favorite lentiviral cloning plasmid 2. Combine LENTI-Smart with your lentiviral cloning plasmid 3. Transfect HEK 293T cells using LENTI-Smart & your lentiviral cloning plasmid 4. Replace with fresh culture medium hours after transfection 5. Harvest supernatant containing lentiviral vectors after hours 6. Titrate lentiviral vectors produced SAFETY CONSIDERATION LENTI-Smart requires Biosafety Level 2 Handle as a potentially biohazardous material under at least Biosafety Level 2 containment and strictly follow all published BSL-2 guidelines with proper waste decontamination. Depending on the transgene used, BSL-3 containment may be required. The lentiviral vector produced with this system may pose a biohazardous risk since it can transduce primary human cells. Furthermore, exercise extra caution when creating lentiviral vectors carrying potentially harmful or toxic genes. For more information about BSL-2 guidelines and lentivirus handling, refer to the document, Biosafety in Microbiological and Biomedical Laboratories, 4th Edition, published by the Centers for Disease Control (CDC). The entire text is available online at Important Handle all lentiviral vectors in compliance with established institutional guidelines. Since safety requirements for use and handling of lentiviral vectors may vary at individual institutions, we recommend consulting the health and safety guidelines and/or officers at your institution prior to use of LENTI-Smart. Always follow microbiological practices, which includes: - wear gloves, safety glasses and laboratory coat when conducting the procedure - always work with lentiviral vectors in a Class II laminar flow hood - perform all procedures carefully to minimize the creation of splashes or aerosols - decontaminate work surfaces at least once a day and after any spill of viable material - decontaminate all cultures, stocks, and other regulated wastes 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. General Information LENTI-Smart is an easy-to-use kit for the production of lentiviral vectors. This system is geared towards users who are familiar with the principles of retrovirus biology and retroviral vectors. We highly recommend that users possess a working knowledge of lentiviral vector production and cell culture techniques. For more information about these topics, refer to the following published reviews: Retrovirus biology and the retroviral replication cycle: see Buchschacher and Wong-Staal 2000, Blood. Retroviral and lentiviral vectors: see Naldini et al., 1998, Curr Opin Biotechnol and Pandya et al. 2001, Expert Opin Biol Ther. 6 7

6 METHODS Growth and Maintenance of HEK 293T cells Growth Medium: DMEM containing 10% FBS, 4.5 g/l glucose, 50 U/ml penicillin, 50 μg/ml streptomycin,1 mm sodium pyruvate, 4 mm L-glutamine and 0.1 mm non-essential amino acids. Cell Culture Plastics Use cell culture plastics which enhance cell adherence, such as Corning CellBIND, as HEK 293T cells may detach during lentiviral production, if not properly bound to plastic. Culturing HEK 293T cells 1. Cells should be grown at 37 C in a humidified 5% CO2 incubator. 2. With a doubling time of less than 24 hours, these cells should be split every 1-2 days when they reach 70-80% confluency. DO NOT allow your cells to reach 90% confluency. For subculturing, detach the cells with trypsin/edta, and split at a ratio of 1:3-1: Prior to use for lentiviral vector production, cells should have undergone at least 2 passages following thawing. 3. Ensure that cells are healthy using Trypan Blue exclusion method (at least 95% viability) at the time of plating. 4. DO NOT use cells after 20 passages. Lentiviral Cloning Plasmid Clone your gene of interest into your favorite lentiviral cloning plasmid. The lentiviral titers vary depending on the size of the sequence between the 3 and 5 LTR, we recommend that the sequences between the LTRs are less that 8 kb. Purify your plasmid DNA using a commercially available midi prep kit. Plasmid DNA for transfection into eukaryotic cells must be very clean and free from contamination, such as phenol and excess sodium chloride. Contamination may increase cell toxicity and residual salt may interfer with the transfection efficiency of LENTI-Smart. We recommend that you: - check the purity of your plasmid DNA. A260/A280 ratio should be perform digestion analysis of plasmid DNA using restriction enzyme(s) of choice - sequence your insert following cloning - test the biological activity of your gene. Positive Control for Lentiviral Vector Production It is good practice to carry out a trial run to highlight any experimental weaknesses. We highly recommend including a positive control lentiviral expression plasmid, such as plv-green, to generate a control lentiviral stock that can be used to help you optimize expression conditions in your target cells. Use 12 mg control plasmid plv-green in place of your lentiviral cloning plasmid to rehydrate LENTI-Smart. Mycoplasma Contamination Figure 1. Healthy 293FT cells at 70% confluence before transfection (magnification 10x). Mycoplasma infection will interfere with lentiviral vector production, therefore, we recommend that you routinely test your cells for mycoplasma contamination using PlasmoTest (see related products, page 4). Normocin can be added to the cell medium to prevent mycoplasma, bacterial and fungal contamination without interfering with the transfection efficiency. Once a myoplasma contamination has been confirmed, it is usually recommended that the infected cell culture be immediately destroyed to prevent the infection from spreading. However, some cell lines are irreplaceable and require an effective eradication treatment, such as Plasmocin, which can be used to treat mycoplasma-contaminated cells. To obtain a working concentration of 12 µg plasmid per 200 µl; - Add 15 µl of sterile water to 15 μg of plv-green. Mix gently until completely dissolved. This will provide a stock solution at 1 μg/μl which can be stored at -20 C. - Pipett 12 μl of the plv-green stock solution (at 1 μg/μl) into a sterile tube and add 188 µl of sterile water. - This 200 μl solution (containing the 12 μg of plv-green) can now be used to rehydate the LENTI-Smart (see page 10, point 3) Thirty-six to forty-eight hours after the transfection, check for GFP expression and fusion of cells in HEK 293T cells using a fluorescent microscope (see page 10, figure 2). The lentiviral stocks can be titered using FACS analysis. 8 9

7 PRODUCTION OF LENTIVIRAL VECTORS USING HEK 293T CELLS Note: This protocol is for one T-75 cm 2 flask (or a 10-cm culture plate), this protocol can be scaled up or down if desired (see table on page 11). 1. Remove LENTI-Smart from -20 C freezer, pre-warm at room temperature for 10 minutes. 2. Prepare a solution containing 12 μg lentiviral cloning plasmid in 200 ml sterile water. 3. Add the 200 μl of the lentiviral cloning plasmid solution to LENTI-Smart. Mix by gently tapping the bottom of the tube. 4. Incubate at room temperature for 15 minutes. 5. Bring total volume to 1 ml, by adding 800 μl of sterile water. Mix well by gently tapping the bottom of the tube. 6. Trypsinize HEK 293T cells, count these cells and assess cell viability using 0.4% (w/v) Trypan Blue exclusion method. 7. Add 1 x 10 7 HEK 293T cells to a T-75 cm 2 flask in 12 ml of pre-warmed growth medium. 8. Place T-75 cm 2 flask upright. Add gently the 1 ml of resuspended LENTI-Smart and lentiviral cloning plasmid directly onto cells. Homogenize thoroughly by pipetting up and down several times. 9. Incubate horizontally the T-75 cm 2 flask containing HEK 293T cells and LENTI-Smart at 37 C, 5% CO After twelve to sixteen hours, discard the culture medium from the HEK 293T cells and replace with 12 ml of pre-warmed fresh growth medium. Incubate the HEK 293T cells at 37 C, 5% CO2. Note: As a visual control, cells should be fused and multinucleated thirty-six to forty-eight hours after transfection, due to the presence of the VSV-G protein (see Figure 2). This morphological change is expected and does not effect the production of lentiviral vectors. If your cells are not fused, refer to troubleshooting section page For optimum lentiviral vector titer, harvest supernatant hours after transfection. Two to three further harvests can be performed every 12 hours. Store the supernatant at 4 C over the collecting period. Note: The color of culture medium should not be yellow, as the medium ph at this point can effect the stability of lentiviral vectors. 12. Pool the collected supernatants, remove cell debris by centrifugation for 5 minutes at 2000 rpm. 13. Filter supernatants using a 0.45 μm PVDF (low protein binding) filter in order to remove viral and cell debris. Note: It is important to perform this filtration step as viral capsid debris can be toxic to your target cells. Optional: DNase I treatment can be carried out to eliminate any residual plasmid for in vivo experiments (see page 12). 14. Titrate the lentiviral supernatant (see pages 13-16) and use the lentiviral vector to transduce target cells, or freeze the stock in aliquots (see point 15). Note: If your cell transduction experiment requires that you use a relatively high MOI, for example in vivo experiments or transduction of stem/primary cells, you may wish to concentrate your lentiviral vectors before proceeding to titration and transduction (see page 12). 15. To freeze the lentiviral supernatant and avoid multiple freeze-thaw cycles, aliquot the supernatant into single-use cryovials. Store tubes at -80 C. No cryoprotectant is required. Supernatant should not be stored for more than 1 year at -80 C. Note: Titers can drop as much as 2-4 fold with each freeze-thaw cycle (Higashikawa & Chang, 2001; Kwon et al., 2003). The volumes of your aliquots depend on your transduction experiments (i.e. as a guideline 5 ml aliquots for transductions in T-25 cm 2 flask; 500 ml aliquots for transductions in a 24-well plate). Figure 2. Fused, multinucleated 293FT cells expressing GFP and VSV-G proteins forty-eight hours after transfection (magnification 40x). The outline of the cell has been traced in white. The intensity of this phenomenon is dependent on the transgene used. Culture Dish/Flask 1 well of a 6-well plate 6-cm culture plate or T-25 cm 2 flask No. of HEK 293T cells (in suspension) Volume of LENTI-Smart Final amount of lentiviral cloning plasmid DNA Final volume of culture medium 1x μl 2 μg 2 ml 3.5x μl 4μg 4 ml T-175 cm 2 flask 2x10 7 2x1ml 2x12 μg 24 ml Numbers of HEK 293T cells, volume of LENTI-Smart and amount of plasmid to be added to each well/dish/flask. For a T-175 cm 2 flask, 2 vials of LENTI-Smart are required. Note: Once LENTI-Smart has been resuspended using a lentiviral cloning plasmid, this solution can be stored at 4 C for 1 month. Do NOT freeze

8 DNAse Treatment of Supernatant containing Lentiviral Vectors 1. Add 1 μl of DNase I (1 mg/ml in PBS 1x) to 1 ml of lentiviral supernatant 2. Add 1 μl of 1M magnesium chloride to 1 ml of lentiviral supernatant 3. Incubate at 37 C for 20 minutes 4. Supernatants can be used directly or purified/concentrated if needed. For long term storage, store supernatant at -80 C in aliquots. Avoid repeated freeze-thaw cycles of the supernatant. Concentration of Lentiviral Vectors A simple method for concentrating lentiviral particles is described below: 1. Centrifuge at RCF 650 g for 30 minutes using a filter, such as Amicon Ultra 100 kda filter. 2. Store supernatant at -80 C in aliquots for 1 year maximum. In order to obtain more concentrated lentiviral supernatant, you may use ultracentrifugation according to the protocol described by Kutner et al. 2009, Nat Protoc. Small Scale Production of Lentiviral Vectors Note: This protocol can be used to test different lentiviral cloning plasmids and produce lentiviral vectors on a small scale. 1. Remove LENTI-Smart from -20 C freezer, pre-warm at room temperature for 10 minutes. 2. Rehydrate LENTI-Smart with 400 μl of sterile water. Leave at room temperature for 15 min. 3. Prepare 3 μl (3 μg) of each lentiviral cloning plasmid at a concentration of 1 μg/μl. 4. Add 100 μl of the resuspended LENTI-Smart to your 3 μg of lentiviral cloning plasmid. Mix by gently tapping the bottom of the tube. Incubate at room temperature for 15 minutes. 5. Trypsinize HEK 293T cells, count these cells and assess cell viability using 0.4% (w/v) Trypan Blue exclusion method. 6. Add 1 x 10 6 HEK 293T cells to one well of a 6-well plate in 2 ml of pre-warmed growth medium. 7. Gently add the LENTI-Smart and lentiviral cloning plasmid directly onto cells. Homogenize thoroughly by pipetting up and down several times. Incubate at 37 C, 5% CO2. 8. After twelve to sixteen hours, discard the culture medium from the HEK 293T cells and replace with 2 ml of pre-warmed fresh growth medium. Incubate the HEK 293T cells at 37 C, 5% CO2. Continue with protocol on page 11, point 11. TITRATION OF LENTIVIRAL VECTORS We recommend that you estimate the titer of the lentiviral vectors within your supernatant before proceeding with transduction experiments for the following reasons: - to ensure that the lentiviral vectors are viable - to normalize batch to batch variations in transduction conditions. A number of factors can influence viral titers including: - the size and nature of your gene of interest. Titers will decrease with increasing size and cytotoxicity of the transgene - the characteristics of the cell line used for titering - the age of your lentiviral stock - the number of freeze-thaw cycles. Titers can drop as much as 2-4 fold with each freeze-thaw cycle - improper storage of your lentiviral stock. Lentiviral stocks should be stored at -80 C in cryovials. p24 ELISA Lentivirus vectors contain major structural proteins, such as the VSV-G envelop protein, the matrix and the viral capsid protein (p24), which is encoded by the gag gene. An ELISA can determine the amount of p24 in the supernatant which is directly proportional to the amount of lentiviral vectors. It must be emphasized that although measurement of p24 (expressed in ng/μl) is a practical way of quantifying lentiviral vectors, the assay does not provide information on the functionality of the produced vector. Moreover this technique tends to overestimate the functional lentiviral titer, as the p24 protein pool that is quantified contains free p24 and p24 from non-functional lentiviral particles. A practical way, to correlate p24 measurements with titer is to produce your lentiviral vectors coding your GOI and in parallel lentiviral vectors coding for GFP. The functional titer of your GFP coding lentiviral vectors, as determined by fluorescence-activated cell sorting (FACS, see page 14), can be used to correlate the p24 value to lentiviral vector content. As a general guideline, 1 ng of p24 is equivalent of TU depending on your lentiviral cloning plasmid. For further information on titration methods, see Geraets et al., 2006 and Tiscornia et al., 2006). Selecting a Cell Line for Titering We strongly recommend the human fibrosarcoma line HT1080 (ATCC, cat no. CCL-121) as the gold standard for titering lentiviral vectors. If you have more than one lentiviral construct, we recommend that you titer all of the lentiviral constructs using the same mammalian cells. The most commonly used cell lines for titering are adherent cells which exhibit a doubling time in the range of hours. For example, HEK 293, HeLa or NIH/3T3 cells can be used to titer your lentiviral vectors

9 FACS Analysis Titration Method 1. Twenty-four hours prior to titration, seed 5 x 10 4 HT1080 cells in 0.25 ml of medium per well of a 48-well plate. 2. On the day of transduction the cells should be 30-50% confluent, trypsinize and count the number of cells in three of the wells. Take the average of these three numbers and use the average for the lentiviral titer calculations (see below). As HT1080 cells grow slowly, normally the numbers of cells per well is approximately 5 x Prepare dilutions of your supernatants in growth medium containing 8 μg/ml of Polybrene. - For non-concentrated supernatant, use 2-fold dilutions, 1/2, 1/4, 1/8, 1/16/ 1/32, 1/64, 1/ For concentrated supernatant, use 10-fold dilutions, 1/10 2, 1/10 3, 1/10 4, 1/10 5,1/10 6,1/ Remove the medium from the wells and add 200 μl of each dilution per well containing the HT1080 cells. 5. Incubate for 4 hours at 37 C, 5% CO2. 6. Remove supernatant and replace it with 500 ml fresh medium. 7. Incubate for 72 hours at 37 C, 5% CO2. 8. Analyze the expression of the fluorescent transgene, such as GFP, by FACS analysis. The following formula is used to calculate the titer: TITER= (F x C x D)/0.2 where: F = frequency of GFP-positive cells (percentage obtained divided by 100) C = average number of cells per well on the day of transduction D = coefficient of lentivirus dilution (e.g. coefficient of dilution is 64 for 1/64 dilution) V = 0.2 ml (volume of inoculum). The titer should be calculated using a percentage of GFP-positive cells within the range of 1-30%. An example for calculating the lentiviral titer is provided. Lentiviral Dilution % GFP positive cells 1/ % 1/ % 1/ % In this example, the 1/64 and 1/128 dilutions are used to calculate the titer, since the percentages of GFP-positive cells fall into the desired range of 1-30%. For this example, the average number of cells per well on the day of transduction is 5 x Thus the calculations for the 1/64 and 1/128 dilutions are as follows: (0.287 x 50,000 x 64)/0.2 = x 10 6 (0.121 x 50,000 x 128)/0.2 = x 10 6 Take the average of these two figures x 10 6 and x The lentiviral titer for this example is 4.2 x 10 6 TU/ml. Note: Viral titers will vary depending on the cell line used for titration and the protocol used. When titering with HT1080 cells and FACS analysis, we generally obtain titers ranging from 1-5 x 10 6 transducing units (TU)/ml (unconcentrated) for integrating vectors. For NIL vectors, the titers are generally 5 to 10-fold lower using the FACS analysis method. If the titer of your concentrated lentiviral stock is less than 1 x10 5 TU/ml for integrating lentiviral vectors (or 1 x 10 4 TU/ml for NIL vectors), we recommend producing a new lentiviral stock. Antibiotic Selection Titration Method This method can be used if your lentiviral vector contains a mammalian selectable marker, such as a gene for antibiotic resistance. Blasticidin, Puromycin and Zeocin are typically used as selection antibiotics in lentiviral vector production. Cells are transduced with serial dilutions of the lentiviral vector stock and then selected for stable transductants using the appropriate antibiotic. Titers are calculated from the number of drug-resistant colonies that develop after selection is completed. In order to titrate using the antibiotic selection method, you must first determine the minimum concentration of antibiotic required to kill your untransduced cell line (i.e. perfom a kill curve experiment). Typically, concentrations ranging from 2-10 μg/ml Blasticidin, 1-10 μg/ml Puromycin or μg/ml Zeocin are sufficient to kill most untransduced mammalian cell lines. We recommend that you test a range of concentrations to ensure that you determine the minimum concentration necessary for your cell line. Due to the limited duration of transgene expression with NIL vectors, antibiotic selection is not feasible for titering NIL vectors. To determine the titer of your integrating lentiviral vector stock using HT1080 cells and antibiotic selection Note: You will need at least one 6-well plate for every lentiviral stock to be titered (one mock well plus five dilutions). 1. Twenty-four hours prior to titration, plate 2 x 10 5 HT1080 cells per well of a 6-well plate, so that they will be 30-50% confluent at the time of transduction. Incubate overnight at 37 C, 5% CO2. 2. Prepare dilutions of your supernatants in growth medium containing 8 μg/ml of Polybrene. - For non-concentrated supernatant, use 2-fold dilutions, 1/2, 1/4, 1/8, 1/16/ 1/32, 1/64, 1/ For concentrated supernatant, use 10-fold dilutions, 1/10 2, 1/10 3, 1/10 4, 1/10 5,1/10 6,1/10 7. For each dilution, dilute the lentiviral stock in complete medium to obtain a final volume of 1 ml. DO NOT VORTEX. 3. Remove the culture medium from your cells. Mix each dilution gently by inversion and add to one well of your cells (total volume = 1 ml). 4. Swirl the plate gently to mix. Incubate at 37 C, 5% CO2. 5. Twenty-four hours later, remove the media containing virus and replace with 2 ml of complete medium. Incubate overnight at 37 C, 5% CO

10 6. Twenty-four hours later, treat cells as follows: -For Blasticidin/Puromycin selection, remove the medium and replace with complete culture medium containing the appropriate amount of Blasticidin/Puromycin to select for stably transduced cells. -For Zeocin selection, remove the medium and wash the cells once with PBS. For each well of cells, trypsinize the cells and replate the entire amount into one 10 cm plate containing complete medium with the appropriate amount of Zeocin to select for stably transduced cells. 7. Replace medium with fresh medium containing antibiotic every 3-4 days. 8. After days of selection (day 14-16), you should see no live cells in the mock well and discrete antibiotic-resistant colonies in one or more of the dilution wells. Remove the medium and wash the cells twice with PBS. 9. Count the colonies and determine the titer of lentiviral stock. GENERAL CONSIDERATIONS FOR TRANSDUCTION Once you have generated a lentiviral stock with a suitable titer, you can transduce the lentiviral vector into the mammalian cells of choice and assay for expression of your gene of interest. To obtain optimal expression of your gene of interest, you will need to transduce the lentiviral vector into the cell line using a suitable multiplicity of infection (MOI). MOI is defined as the number of transduction units per cell and generally correlates with the number of integration events and as a result, with the expression of your gene of interest. MOI = (T xv)/n, where T is the titer of your lentiviral vector stock, V is the volume of lentiviral vectors in ml, and N is the number of cells to be transduced. The MOI needed to transduce your cells efficiently depends on the cell line and titration method used. Typically, expression levels increase linearly as the MOI increases. If you are transducing your lentiviral construct for the first time, we recommend using a range of MOI (e.g. 0, 0.5, 1, 2, 5, 10) to obtain the optimal expression of your transgene. For further information, see Zhang et al. 2004, Genet Vaccines. An example of colony counts in a 6-well plate; Mock: no colonies 10-4 dilution: confluent, undeterminable 10-2 dilution: confluent, undeterminable 10-5 dilution: dilution: confluent, undeterminable 10-6 dilution: 6 TITER= No. of colonies x coefficient of dilution. Thus, the titer for this example is 4.6 x 10 6 TU/ml (i.e. average of 32 x 10 5 and 6 x 10 6 ). qrt-pcr Titration Method Lentiviral vector titration performed by qrt-pcr quantifies lentiviral mrna copies following stable transduction in cell culture. Conditions for titering integrating lentiviral stocks by qrt-pcr have been described (Lizee et al., 2003; Geraets et al., 2006; Kutner et al., 2009). TRANSDUCTION PROCEDURE Note: This protocol is a guideline for use with mammalian cells. 1. Plate mammalian cells of choice in complete medium as appropriate for your cells. 2. Calculate the amount of lentiviral vectors required to transduce your cells at the appropriate MOI. An example is given where 5x10 5 cells/ml in one well of 24-well plate in 500 ml of medium are to be transduced at MOI 5 using a lentiviral vector stock with a titer of 5x10 8 TU/ml. Volume of lentiviral vector required = MOI x number of cells to be transduced/lentiviral titer =(5 x 5 x 10 5 )/5 x 10 8 = ml. Add Polybrene (if desired) to a final concentration up to 8 μg/ml. 3. Discard the medium and add fresh medium. Add the lentiviral vectors gently directly onto the cells. Swirl the plate gently to mix. Incubate overnight at 37 C, 5% CO2. Note: Serum-free medium is recommended for the transduction of primary cells. Immortalized cell lines, however, can be transduced in the complete medium. Optional, if your cells are difficult to transduce, we recommend adding a second dose of lentiviral vectors after the overnight incubation. 4. The following day (Day 2), discard the medium containing the lentiviral vector, wash cells twice with PBS (1x) and replace with fresh, complete culture medium. Incubate at 37 C, 5% CO2. 5. Generally, transgene expression is optimal seventy-two hours after transduction. Note: Transduction with integrating lentiviral vectors, results in stable expression of the transgene. However, transduction with NIL vectors results in transient expression of the transgene. Following transduction with NIL vectors, transgene expression is normally between 7-15 days, although the duration of transgene expression is dependent on the rate of cell division (i.e. the faster your transduced cells divide, the shorter the duration of transgene expression). In quiescent cells, such as neurons, NIL transduction will result in long-term expression of your transgene

11 Problem Reason Solution Problem Reason Solution LENTI-Smart precipitates upon rehydration. Low lentiviral vector titer. Residual salt present in your lentiviral cloning plasmid. Used poor quality cloning plasmid DNA (i.e. plasmid DNA from a mini-prep). Use a midi-prep DNA isolation kit to prepare plasmid DNA. Check the purity of plasmid DNA. A260/A280 ratio should be Use a midi-prep DNA isolation kit to prepare plasmid DNA. Check the purity and perform a restriction digest of your plasmid. If the digests do not correspond with the expected sizes, sequence your plasmid or test for reporter gene activity. Low lentiviral vector titer (continued). Gene of interest is too large. Poor choice of titering cell line. Poor quality culture medium. Poor quality fetal calf serum. Lentiviral vector titers generally decrease as the size of the insert increases. Inserts larger than 8 kb are not recommended. Use HT1080 cells. Use good quality cell culture medium, such as Gibco DMEM cat. code Use good quality fetal bovine serum (FBS), such as Thermo Scientific HyClone, cat. code SH Unhealthy HEK 293T cells which exhibit low viability. Mycoplasma contamination. Too few HEK 293T Cells. Lentiviral vector supernatant harvested too early. Use healthy HEK 293T cells. Do not allow cells to reach 90% confluency. Do not use after 20 passages. HEK 293T cells have a doubling time of less than 24 hours, these cells should be split every 1-2 days when they reach 70-80% confluency. Test cells for mycoplasma contamination using PlasmoTest. Treat a mycoplasma contamination with Plasmocin. Normocin can be added to the cell medium to prevent mycoplasma, bacterial and fungal contamination, without interfering with transfection. Add 1 x 10 7 HEK 293T cells to a T-75 cm 2 flask prior to transfection. Collect lentiviral vector supernatant 48 hours post transfection. Titer indeterminable. No expression of the gene of interest following transduction in mammalian cells. HEK 293T Cells are not fused following transfection i.e. during lentiviral vector production. Lentiviral vector supernatant not diluted sufficiently. Promoter silencing. lentiviral vector stocks stored incorrectly. Cells not expressing the VSV-G gene. Titer lentiviral vectors using a wider range of serial dilutions (e.g to 10-8 ). Lentiviral constructs may integrate into a chromosomal region that silences the CMV promoter. Do not freeze/thaw viral supernatant more than 3 times. Use healthy HEK 293T cells (see page 8). Test for mycoplasma contamination (see page 8). Use good quality culture medium and FCS. Check the purity of DNA plasmid, as residual salt and contamination can interfere with transfection efficiency. Repeat experiment using control plasmid coding for the GFP gene. Lentiviral vector supernatant frozen and thawed too many times. Do not freeze/thaw lentiviral vector supernatant more than 3 times. Poor expression of the gene of interest. Multiplicity of infection (MOI) too low. Transduce your lentiviral vectors into cells using a higher MOI. Cells do not adhere to the surface of the cell culture plastics. Gene of interest is toxic. We recommend the use of Corning CellBIND plastics. Cells harvested too soon after transduction. Gene of interest is toxic. Do not harvest cells until at least hours after transduction to allow expressed protein to accumulate in transduced cells

12 EM7 Zeo CMV Ori Gag pan Rev Tat plv-help (8551 bp) RRE Rev Tat Pol Feature Benefit Feature Benefit CMV enhancer/promoter Allows high-level expression of VSV-G in mammalian cells. HIV-1 gag coding sequence Encodes the encodes the membrane associated and capsid proteins. Rabbit beta-globin intron VSV-G glycoprotein (VSV-G) Rabbit beta-globin polyadenylation signal Ampicillin (AmpR) resistance gene Enhances expression of VSV-G in mammalian cells. Encodes the envelop G glycoprotein from Vesicular Stomatitis Virus to allow production of a pseudotyped retrovirus with a broad host range. Allows efficient transcription termination and polyadenylation of mrna. Allows selection of the plasmid in E.coli. HIV-1 pol coding sequence HIV-1 rev coding sequence Encodes the viral replication enzymes, reverse transcriptase and integrase, required for replication and integration of the lentivirus. The plv-help-nil D64 contains a single nucleotide mutation within the pol coding sequence (D64V) resulting in the non-functional integrase enzyme and the production of NIL vectors Encodes the Rev protein that interacts with the RRE to induce Gag and Pol expression, and on the expression vector to promote the nuclear export of the unspliced viral RNA for packaging into viral particles. HIV-1 Rev response element (RRE) Permits Rev-dependent expression of the gag and pol genes 20 21

13 USE RESTRICTIONS LENTI-Smart is distributed for research purposes only. InvivoGen warrants that this product will perform according to established product specifications. It is sold with the understanding that the purchaser will determine if the product is suitable for his or her application. InvivoGen shall not be liable for any damages or injury to persons or property arising from the purchase or use of the product. Feature CMV enhancer/promoter HIV-1 truncated 5 LTR HIV-1 psi packaging signal HIV-1 Rev response element (RRE) CMV enhancer and Ferritin Light promoter Green fluoresence protein (GFP) DU3/HIV-1 truncated 3 LTR Benefit Allows Tat-independent production of lentiviral mrna Permits viral packaging and reverse transcription of the lentiviral mrna Allows lentiviral packaging Permits Rev-dependent nuclear export of unspliced lentiviral mrna Permits high-level expression of GFP in a broad range of mammalian cells Encodes a green fluorescent protein that absorbs blue light (at 480 nm) and emits green light (at 505 nm). Allows viral packaging but self-inactivates the 5 LTR for biosafety purposes. 22 Limited Use License The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes. Product covered by patent pending (Patent application number 61/223,530). Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. If the purchaser is not willing to accept the limitations of this limited use statement, InvivoGen is willing to accept return of the product with a full refund. For information on purchasing a license to this product for purposes other than research, contact InvivoGen, 3950 Sorrento Valley Blvd. Suite 100, San Diego California Tel: Fax: REFERENCES Apolonia L. et al., Stable gene transfer to muscle using non-integrating lentiviral vectors. Mol. Therapy 15, Blomer U. et al., Highly efficient and sustained gene transfer in adult neurons with a lentivirus vector. J Virol. 71; Buchschacher G.L. & Wong-Staal F., Development of lentiviral vectors for gene therapy for human diseases. Blood 95; Burns J.C. et al., Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc Natl Acad Sci USA 90; Geraerts M et al., Comparison of lentiviral vector titration methods. BMC Titration. 6;34. Higashikawa F. & Chang L., Kinetic analyses of stability of simple and complex retroviral vectors. Virology 280; Kutner R.H. et al., Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors. Nat Protoc 4; Kwon Y.J. et al., Determination of infectious retrovirus ocncentration from colony-forming assay with quantitative analysis. J Virol. 77; Leavitt AD. et al., Human immunodeficiency virus type 1 integrase mutants retain in vitro integrase activity yet fail to integrate viral DNA efficiently during infection. J Virol. 70(2): Lizee G. et al., Real-time quantitative reverse transcriptase-polymerase chain reaction as a method for determining lentiviral vector titers and measuring transgene expression. Hum Gene Ther 14: Naldini L. et al, In vivo gene delivery and stable transduction of non-dividing cells by a lentiviral vector. Science 272; Naldini L. et al, Lentiviruses as gene transfer agents for delivery to non-dividing cells. Curr Opin Biotechnol.9; Negri D.R.M. et al., Successful immunization with a single injection of ono-integrating lentiviral vector. Mol Ther 15, Nightingale S.J. et al., Transient gene expression by nonintegrating lentiviral vectors. Mol Ther 13, Pandya S. et al., Lentivirus and foamy virus vectors:novel gene therapy tools. Expert Opin Biol Ther. 1; Tiscornia et al., Production and purification of lentiviral vectors. Nat Protoc 1; Verma I.M. & Somia N., Gene therapy- promises, problems and propects. Nature 389, Yanez-Munoz, R.J., et al., Effective gene therapy with nonintegrating lentiviral vectors. Nat Med 12, Zhang B. et al., The significance of controlled conditions in lentiviral vector titration and in the use of multiplicity of infection (MOI) for predicting gene transfer events. Genet Vaccines 2; 6. 23