Name: Lentivirus Production (Video) | JoVE Journal
Uploaded: 2009-10-02T21:00:00.000+00:00
Duration: 11 min 42 s
Description: Learn lentivirus production using 293T cell transfection, ultracentrifugation concentration, and fluorescence-based titer determination. This reliable protocol enables efficient gene delivery for research and therapeutic applications.

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5 minutes

Sandler Asthma Basic Research Center (SABRE) 
Sandler Foundation

Part 1: Transfection of HEK 293 Cells To Produce Lentiviruses
*24 hours before transfection, plate 2.5 X 106 cells in a 10cm dish for a confluency of 50-70% the next day.
<ol>
 <li>Start this protocol by preparing the DNA with which one will later transfect one's cells to make virus. First, dilute FuGENE 6 Transfection Reagent Roche, a lipid reagent that make cells take up DNA. In a 1.5 ml tube, pipette 30μl FuGENE 6 into 600ul serum-free medium (SFDMEM). Be careful to not let FuGENE touch the side of the tube as it is delivered into the medium. Let this FuGENE and SFDMEM mix and incubate at room temperature for 5 minutes. </li>
 <li>In the cap of the tube, mix 4 μg lentiviral plasmid with 4 μg each 3rd generation viral packaging vectors (pMDL, pRSV and pVSV-G). The lentiviral plasmid contains the DNA the virus will insert into the genome of every cell it infects, while the packaging vectors contain genes for all the other proteins required to make a lentivirus. </li>
 <li>Close the lid and mix by inverting the tube a few times.</li>
 <li>Incubate 15-20 minutes at room temperature.</li>
 <li>Pipette entire content of the tube into 293 plate. Mix gently by tilting plate back and forth.</li>
 <li>Return the cells to the incubator and allow viral production to continue for 48-96 hours before harvest. </li>
 <li>During incubation, the 293 cells transcribe the DNA of interest from the lentiviral plasmid, creating an RNA copy of the lentiviral construct. They also transcribe and translate the genes in the packaging vectors into viral proteins. These proteins proceed to make viruses containing the RNA version of your lentiviral construct, which the virus will reverse transcribe and insert into the genomes of all the cells it infects.</li>
</ol>
Part 2: Lentiviral Harvest
<ol>
 <li>After incubation period, take cells out of the incubator and use a disposable, 10ml syringe to remove the supernatant, which now contains the virus. There will be about 10ml of supernatant.</li>
 <li>Attach a 0.45μM syringe filter to the tip and filter the virus into a Beckman Ultracentrifuge tube. The filter allows only viruses, and not cells to pass through. </li>
 <li>Before discarding, incubate all virus-producing cells, culture plates, syringes and filters in 10% bleach for 45 minutes.</li>
</ol>
Part 3: Lentiviral Concentration via Ultracentrifugation
<ol>
 <li>Load centrifuge tubes into SW-41Ti or SW-28 rotor buckets.</li>
 <li>Use serum-free DMEM to balance all virus-containing tubes to within 0.2g.</li>
 <li>Seal shut rotor buckets and hook them onto rotor before placing rotor inside the ultracentrifuge</li>
 <li>Spin the virus for 90 minutes in an ultracentrifuge at 4°C at 25,000 rpm in an SW-41Ti rotor or 24,000rpm in an SW-28 rotor.</li>
 <li>In a tissue culture hood, invert tubes upside down to pour supernatant into a container containing 10% bleach. </li>
 <li>Use a Kimwipe to absorb excess liquid around the pellet and invert the tube in a convenient rack for no more than five minutes.</li>
 <li>To re-suspend the viral pellet, use a filter tip to add 100 μL sterile PBS to the tube, then pipette up and down about 20 times.</li>
 <li>Leave the virus at 4°C overnight to complete re-suspension. One can store viral preps at 4 degrees for about 1 week and at -80
 degrees for up to one year.  Remember to treat all empty tubes with 10% bleach before discarding. </li>
 <li>If one intends to use the viral prep for longer than the 1 week, make minimal aliquots of 5-20μl for future experiments and 1 aliquot of 3 to 5μl for the titration. Freeze and store the aliquots at minus 80. </li>
</ol>
Part 4: Lentiviral Titration Using Flow Cytometry
<ol>
 <li>The next step is to determine the viral titer by flow cytometry. This method only works in cases where the lentiviral plasmid contains a fluorescent reporter gene. The idea is that when the virus infects a cell, it introduces the lentiviral plasmid DNA into that cell’s genome. The infected cell will then transcribe and translate the genes you included in the lentiviral plasmid, and if you included a fluorescent reporter gene, infected cells will fluoresce. The level of fluorescence represents the potency, or titer, of the virus. </li>
 <li>For each viral stock to be titered, dilute 3 μl of concentrated virus into 1.5 ml of complete media (DMEM, pen-strep, glutamine, FBS).</li>
 <li>In wells 1-6 in a single row of a 96 well plate, perform the following serial dilutions: to all 6 wells add 100 μl of complete DMEM media. To the first well add 100μl of media with no virus (this is your unstained control), to the second well add 100 μl of the diluted virus (which is equivalent to1 ml of undiluted virus), to the third well add 100 μl of the mix from the second well, to the fourth well add 100 ml from the 3rd well, to the fifth well add 100μl from the 4th well, and to the sixth well add 100 μl from the 5th well. Finally take 100ul out of the 6th well and discard in bleach. A serial dilution was performed where each well has half the volume of virus compared to the one before it.</li>
 <li>Bring the total volume in all wells to 200μl. Note that these are only suggested dilutions, which should work well for titering most concentrated viruses. If the virus is low in titer, or unconcentrated one may need to adjust the dilutions.</li>
 <li>Treat any unused diluted virus with 10% bleach for 45 minutes before discarding into a biohazard waste container.</li>
 <li>Add ~15,000 healthy, actively dividing 293 cells to each of the 6 wells. A single 96-well plate can be used to titer 16 viral preps. </li>
 <li>Return cells to 37 degree incubator with 5% CO2 and allow infection to proceed for at least 48 hours.</li>
 <li>After the incubation, the cells are analyzed for reporter expression (in our case fluorescence) with a flow cytometer. Viral particles are quantitated by the percent of fluorescent cells per well. </li>
</ol>
Calculate the number of infectious units per ml with the following formula:
<img src="/files/ftp_upload/1499/1499eq1.jpg" alt="Cell quantification formula, infection reporter analysis, vector volume calculation, scientific computation." data-altupdatedat="1787630408000" data-alt="figure 1">
Part 5: Representative Results:
After a 48-hour incubation period post transfection, the plate of 293 cells should be at around 90% fluorescent. A high percentage of fluorescent cells is an indication of a high percentage of virus producing cells.
After centrifugation, the viral pellet is barely visible when resuspending. This is normal as the pellet is clear and very small.
When interpreting flow cytometer results, note that one can only achieve accurate titer calculations when infected (fluorescent) cells have received no more than one viral integration per cell.  In order to ensure that this is the case, use cells with &lt; 15 % infection rate for calculations. Cells with higher infection rates have likely received multiple viral integrants per cell. The dilutions suggested in step 2 should yield several samples within this range of infection. Ideally, use several samples to generate a titration plot (linear line) from which you can calculate the final titer, but you can calculate titer using a single sample, if necessary. One can expect titer of 1.0 x 107 TU/ml for un-concentrated virus and 1.0 x 108 TU/ml for concentrated virus.

<ol><li>Reiser, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+and+concentration+of+pseudotyped+HIV-1-based+gene+transfer+vectors.%5BTitle%5D)+AND+%22Gene+Therapy%22%5BJournal%5D)">Production and concentration of pseudotyped HIV-1-based gene transfer vectors.</a> Gene Therapy. , 72-7211 (2000).</li>
<li>Dull, T., Zufferey, R., Kelly, M., Mandel, R. J., Nguyen, M., Trono, D., Naldini, L. J Virol. 72 (11), 8463-8471 (1998).</li>
</ol>

<table><tbody><tr><td>DMEM HEK 293T cells Penicillin-streptomycin Glutamine Phosphate-buffered saline FBS</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>FuGENE 6 transfection reagent pSico mCherry pMDL pRSV pVSVG</td><td>Roche Group</td><td></td><td></td></tr><tr><td>Bleach</td><td>Clorox</td><td></td><td></td></tr><tr><td>10-cm cell culture dishes 96 well cell culture dishes </td><td>Falcon BD</td><td>353003</td><td></td></tr><tr><td>Multichannel pipette</td><td>Rainin</td><td></td><td></td></tr><tr><td>Filtered pipette tips</td><td>Rainin</td><td></td><td></td></tr><tr><td>1.5ml Eppendorf tubes Syringe Syinge filter (0.45-&amp;mu;m)</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Ultracentrifuge tubes</td><td>Beckman Coulter Inc.</td><td></td><td></td></tr><tr><td>Tissue culture incubator at 5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Coulter</td><td></td><td></td></tr><tr><td>Beckman SW41T.I rotor</td><td></td><td></td><td></td></tr><tr><td>Beckman ultracentrifuge</td><td>Beckman Coulter Inc.</td><td></td><td></td></tr><tr><td>Fluorescent microscope</td><td>Coulter</td><td></td><td></td></tr><tr><td>FACS Array</td><td>Beckman Coulter Inc.</td><td></td><td></td></tr></tbody></table>

lentivirus production

Lentivirus Production

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are silenced at the post-transcriptional state. Short hairpin RNAs, precursors to siRNA, can be expressed using lentivirus, allowing for RNAi in a variety of cell types. Lentiviruses, such as the Human Immunodeficiency Virus, are capable to infecting both dividing and non-dividing cells. We will describe a procedure which to package lentiviruses. Packaging refers to the preparation of competent virus from DNA vectors. Lentiviral vector production systems are based on a 'split' system, where the natural viral genome has been split into individual helper plasmid constructs. This splitting of the different viral elements into four separate vectors diminishes the risk of creating a replication-capable virus by adventitious recombination of the lentiviral genome. Here, a vector containing the shRNA of interest and three packaging vectors (p-VSVG, pRSV, pMDL) are transiently transfected into human 293 cells. After at least a 48-hour incubation period, the virus containing supernatant is harvested and concentrated. Finally, virus titer is determined by reporter (fluorescent) expression with a flow cytometer.

To make lentiviruses, DNA vectors are transfected into human 293 cells. After harvest and concentrating the supernatant, virus titer is determined by fluorescence expression with a flow cytometer.

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are ...

This procedure begins with transecting 2 9 3 T cells with lentivirus, plasmid and viral packaging vectors, the viral supernatant is harvested after at least a 48 hour incubation period. The snat is filtered to be concentrated with an ultracentrifuge after ultracentrifugation. The viral pellet is resuspended with PBS to obtain virus titer, first dilute concentrated virus in complete media, then perform serial dilution to infect cells with lentivirus.Hi, I'm Shein Wong from the laboratory of Michael McManus at the Diabetes Center in the University of California San Francisco. I'm Michael McManus. Today we'll show you a procedure for the production of lentiviruses.We use this procedure in the laboratory to make lentiviruses for RNA interference. So let's get started. To begin this protocol, prepare the DNA with which to transfect the 2 9 3 T cells.First dilute FU gene six transfection reagent in a 1.5 milliliter tube by mixing 30 microliters Fuji six with 600 microliters serum free medium. Be careful to not let Fuji touch the side of the tube as you deliver it into the medium incubate room Temperature for five minutes. Next, in the cap of the tube, mix four micrograms lentiviral plasmid with 1.33 micrograms.Each third generation viral packaging vectors. The lentiviral plasmid contains the DNA. The virus will insert into the genome of every cell infects while the packaging vectors contain genes for all the other proteins required to make a lentil virus close the lid and mix by inverting the tube a few times.Incubate the DNA and transfection reagent for 15 to 20 minutes at room temperature. Now it is time to transfect 2 9 3 cells with the DNA you have prepared use cells that you have plated 24 hours in advance and that have reached 50 to 70%co fluency in the tissue culture hood pipette all of the prepared DNA onto the 2 9 3 plate mixed gently by tilting the plate back and forth, return the cells to the incubator and allow viral production to continue for 48 to 96 hours before harvest. During incubation, the 2 9 3 cells produce lentiviruses whose genomes contain an RNA copy of your DNA of interest.Now it is time to collect the virus. Take your cells out of the incubator. Have ready in the tissue culture hood, a 0.4 or five micron syringe filter, a disposable 10 milliliter syringe and an UltraClear Beckman centrifuge tube.Use the disposable syringe to remove the supernatant, which now contains the virus. Attach a 0.45 micron syringe filter to the tip and filter the virus into an UltraClear Beckman centrifuge tube. The filter allows only viruses and not cells to pass through before discarding, incubate all virus producing cells.Culture plates, syringes and filters in 10%bleach for 45 minutes. Now that you have collected and filtered the virus, it is time to concentrate the virus by ultracentrifugation first. Use serum free DMEM to balance all virus containing tubes to within 0.2 grams of each other.Next, load the balanced centrifuge tubes into SW 41 TI or SW 28 rotor buckets. Seal the rotor buckets shut and hook them onto the rotor. Then place the rotor inside the ultracentrifuge.Spin the virus for about 90 minutes at four degrees Celsius at 25, 000 RPM in an SW 41 TI rotor, or 24, 000 RPM in an SW 28 rotor. After centrifugation, return to the tissue culture hood and invert the tube to pour the SNAT into a container containing 10%bleach. Use a Kim wipe to absorb excess liquid around the pellet and invert the tube in a convenient rack for no more than five minutes until you are ready to resuspend the pellet.To resuspend the viral pellet, use a filter tip to add 100 microliters of sterile pre-filtered PBS to your tube. Then pipette up and down about 20 times. Leave the virus at four degrees overnight to complete resuspension.You can store viral preps at four degrees for about one week and at minus 80 degrees for up to one year. Remember to treat all empty tubes with 10%bleach before discarding. If you intend to use the viral prep for longer than the one week, it will keep in the fridge.Make minimal aliquots of five to 20 microliters for future experiments and one aliquot of three to five microliters for the titration freeze and store the aliquots at approximately minus 80. The next step is to determine the viral titer. If you have included a fluorescent reporter gene in your lentiviral plasmid infected cells will fluoresce and you can determine the viral titer using flow cytometry.To begin, dilute three microliters of concentrated virus into 1.5 milliliters of complete medium in wells, one to six. In a single row of a 96 well plate. Perform the following serial dilutions to all six wells at 100 microliters of complete DM.To the first well add 100 microliters of medium with no virus.This is your unstained control. To the second, well add 100 microliters of the diluted virus to the third. Well add 100 microliters of the mix from the second well to the fourth.Well add 100 microliters from the third well to the fifth. Well add 100 microliters from the fourth well and to the sixth. Well add 100 microliters from the fifth.Well finally take 100 microliters out of the sixth well and discard and bleach bring the total volume in all of the wells to 200 microliters with DMEM. With each dilution, the viral concentration decreases by half. Note that these are only suggested dilutions, which should work well for titering most concentrated viruses.If your virus is low in titer or unconcentrated, you may need to adjust your dilutions. Treat any unused diluted virus with 10%bleach for 45 minutes before discarding into a biohazard waste container. Add about 15, 000 healthy actively dividing 2 9 3 T cells to each of the six wells.You can use a single 96 well plate to titer 16 viral preps, top off wells to 200 microliters, return cells to the incubator and allow infection to proceed for at least 48 hours. After the incubation, analyze the cell fluorescence using a flow cytometer. You can use the percentage of fluorescent cells per well to determine the titer or number of infectious viral particles per milliliter of virus added.Calculate the number of infectious units per milliliter with the following formula when interpreting flow. Cytometer results. Note that one can only achieve accurate titer calculations when infected cells have received no more than one viral integration per cell.In order to ensure that this is the case, use cells with less than 15%infection rate for calculations. Cells with higher infection rates will likely have received multiple viral integr per cell. The dilution suggested in step two should yield several samples within this range of infection.Ideally use several samples to generate a titration plot from which you can calculate the final titer. The calculated formula from the fitted linear line can be used to calculate the viral titer, but you can calculate titer using a single sample if necessary. One can expect titer of one times 10 to the seventh transducing units per milliliter for unconcentrated virus, and one times 10 to the eighth transducing units per milliliter for concentrated virus.We've just shown you how to produce lentiviruses when doing this procedure. It's important to adhere to all biosafety regulations and dispose of waste properly. So that's it.Thanks for watching and good luck with your experiments.

Research

JoVE Journal

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Lentiviral expression vectors are the most effective vehicles for stably expressing different effector molecules or reporter constructs in dividing and non-dividing mammalian cells and whole organisms.  Here we provide a protocol on how to package lentivector expression constructs in pseudoviral particles and to transduce target cells using the pseudoviral particles.

Packaging HIV- or FIV-based Lentivector Expression Constructs &amp; Transduction of VSV-G Pseudotyped Viral Particles

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A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

Here we present protocols that offer a flexible and strategic foundation for virally manipulating oligodendrocyte precursor cells to overexpress proteins of interest in order to specifically interrogate their role in oligodendrocytes via the in vitro model of central nervous system myelination.

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We describe the production strategy of integrase-deficient lentiviral vectors (IDLVs) as vehicles for delivering CRISPR/Cas9 to cells. With an ability to mediate quick and robust gene editing in cells, IDLVs present a safer and equally effective vector platform for gene delivery compared to integrase-competent vectors.

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

Here, we present a protocol to promote transgene integration and production of founder transgenic mice with high efficacy by a simple injection of a lentiviral vector in the perivitelline space of a fertilized oocyte.

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

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Lentiviral Vector Preparation for Efficient Gene and MicroRNA Modulation of Peritoneal Cavity Tissue&#45;Resident Macrophages In Vivo in Mice

Polyglutamine spinocerebellar ataxias are caused by CAG expansions producing aggregation-prone proteins. This protocol describes vector production, stereotaxic delivery, and analysis of inclusions and neuronal marker loss, providing a controlled platform to investigate mechanisms and evaluate targeted therapies for neurodegenerative diseases.

Lentiviral-Induced Striatal Pathology as a Preclinical Model of Polyglutamine Spinocerebellar Ataxias

Here, we describe a method for cell-based visualization, subcellular localization, and quantitative analysis of poly(ADP-ribose) (PAR) enriched foci in fixed mammalian cells. This assay enables the quantification of sites of DNA damage-induced PARP activation, including that associated with base excision repair or single-strand break repair, in the nuclei of genotoxin-exposed cells.

Visualization and Quantitative Analysis of Genotoxin-Induced PARP1/PARP2 Activation in Cells Using a Fluorescent Fusion Protein-Based Reporter

Learn lentivirus production using 293T cell transfection, ultracentrifugation concentration, and fluorescence-based titer determination. This reliable protocol enables efficient gene delivery for research and therapeutic applications.

Lentivirus Production

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