Introduction

The ViraPower™ Lentiviral Expression System allows creation of a replication - incompetent, HIV-1-based lentivirus that is used to deliver and express your gene of interest in either dividing or non-dividing mammalian cells. The major components of the system include:

  • An expression plasmid containing the gene of interest under the control of a choice of promoters, and elements that allow packaging of the construct into virions.
  • An optimized mix of the three packaging plasmids (pLP1, pLP2, and pLP/VSVG) that supply the structural and replication proteins in trans that are required to produce the lentivirus
  • The 293FT cell line, which allows production of lentivirus following cotransfection of the expression plasmid and the plasmids in the packaging mix
  • Control expression plasmid to optimize virus production and cell transduction, containing either:

  1. The lacZ gene which when packaged into virions and transduced into a mammalian cell line, expresses β-galactosidase (included with each expression vector), or
  2. The Emerald Green Fluorescent Protein (EmGFP) gene which when packaged into virions and transduced into a mammalian cell line, expresses EmGFP (available separately; see ordering information below)

For more information on expression vectors and the corresponding positive control vectors, refer to the manual for the specific expression or control vector you are using.

Advantages of the System

Use of the ViraPower™ Lentiviral Expression System to facilitate lentiviral-based expression of the gene of interest provides the following advantages:

  • Generates an HIV-1-based lentivirus that effectively transduces both dividing and non-dividing mammalian cells, thus broadening the potential applications beyond those of traditional Moloney Leukemia Virus (MoMLV)-based retroviral systems (Naldini, 1998)
  • Efficiently delivers the gene of interest to mammalian cells in culture or in vivo (Dull et al., 1998)
  • Provides stable, long-term expression of a target gene beyond that offered by traditional adenoviral-based
  • Produces a pseudotyped virus with a broadened host range (Yee et al., 1994)
  • Includes multiple features designed to enhance the biosafety of the system

This page provides an overview of the ViraPower™ Lentiviral Expression System and provides instructions and guidelines to:

  1. Co-transfect the pLenti-based expression vector and the ViraPower™ Packaging Mix into the 293FT Cell Line to produce a lentiviral stock.
  2. Titer the lentiviral stock.
  3. Use the lentiviral stock to transduce your mammalian cell line of choice.
  4. Assay for “transient” expression of your recombinant protein, or
  5. Generate a stably transduced cell line, if desired.

For details and instructions to generate your expression vector, refer to the manual for the pLenti vector you are using. For instructions to culture and maintain the 293FT producer cell line, refer to the 293FT Cell Line manual. These manuals are supplied with the ViraPower™ Lentiviral Expression Kits, and are also available for downloading from our web site at www.invitrogen.com or by contacting Technical Support.

Components of the ViraPower™ Lentiviral Expression System


The ViraPower™ Lentiviral Expression System facilitates highly efficient, in vitro or in vivo delivery of a target gene to dividing and non-dividing mammalian cells using a replication-incompetent lentivirus. Based on the lentikat™ system developed by Cell Genesys (Dull et al., 1998), the ViraPower™ Lentiviral Expression System possesses features which enhance its biosafety while allowing high-level gene expression in a wider range of cell types than traditional retroviral systems. The System includes the following major components:

  • A pLenti-based expression vector into which the gene of interest will be cloned. The vector also contains the elements required to allow packaging of the expression construct into virions (e.g., 5′ and 3′ LTRs, Ψ packaging signal). For more information about the pLenti expression vectors, refer to the manual for the specific vector you are using.
  • The ViraPower™ Packaging Mix that contains an optimized mixture of the three packaging plasmids, pLP1, pLP2, and pLP/VSVG. These plasmids supply the helper functions as well as structural and replication proteins in trans required to produce the lentivirus.
  • An optimized 293FT producer cell line that stably expresses the SV40 large T antigen under the control of the human CMV promoter and facilitates optimal production of virus. For more information about the 293FT Cell Line, refer to the 293FT Cell Line manual.

You will cotransfect the ViraPower™ Packaging Mix and the pLenti vector containing your gene of interest into 293FT cells to produce a replication incompetent lentivirus, which will be used to transduce a mammalian cell line  of interest.

How Lentivirus Works

Once the lentivirus enters the target cell, the viral RNA is reverse-transcribed, actively imported into the nucleus (Lewis & Emerman, 1994; Naldini, 1999), and stably integrated into the host genome (Buchschacher & Wong-Staal, 2000; Luciw, 1996). After the lentiviral construct has integrated into the genome, you may assay for transient expression of your recombinant protein or use antibiotic selection to generate a stable cell line for long-term expression studies.

VSV Envelope Glycoprotein

Most retroviral vectors are limited in their usefulness as gene delivery vehicles by their restricted tropism and generally low titers. In the ViraPower™ Lentiviral Expression System, this limitation has been overcome by use of the G glycoprotein gene from Vesicular Stomatitis Virus (VSV-G) as a pseudotyping envelope, thus allowing production of a high titer lentiviral vector with a significantly broadened host cell range (Burns et al., 1993; Emi et al., 1991; Yee et al., 1994).
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Ordering Information

Sku Name Size Price Qty
K495000 ViraPower™ Lentiviral Directional TOPO® Expression Kit 1 kit USD 1,932.00
K496000 ViraPower™ Lentiviral Gateway® Expression Kit 1 kit USD 1,698.00
K36720 ViraPower™ II Lentiviral Gateway® Expression System 1 kit USD 1,736.00
K37020 ViraPower™ II Lentiviral C-Lumio™ Gateway® Expression System 1 kit USD 1,754.00
K37120 ViraPower™ II Lentiviral N-Lumio™ Gateway® Expression System 1 kit USD 1,754.00
K497000 ViraPower™ Bsd Lentiviral Support Kit 20 reactions USD 968.00
K497500 ViraPower™ Lentiviral Packaging Mix 60 reactions USD 1,938.00
K498000 ViraPower™ Zeo Lentiviral Gateway® Expression Kit 1 kit USD 1,698.00
K498500 ViraPower™ Zeo Lentiviral Support Kit 20 reactions USD 978.00
K499000 ViraPower™ UbC Lentiviral Gateway® Expression Kit 1 kit USD 1,698.00

Biosafety Features of the System

 

Introduction

The ViraPower™ Lentiviral Expression System is a third-generation system based on lentiviral vectors developed by Dull et al., 1998. This third-generation lentiviral system includes a significant number of safety features designed to enhance its biosafety and to minimize its relation to the wild-type, human HIV-1 virus. These safety features are discussed below.

Biosafety Features of the ViraPower™ Lentiviral System

The ViraPower™ Lentiviral Expression System includes the following key safety features:

  • The pLenti expression vector contains a deletion in the 3′ LTR (ΔU3) that does not affect generation of the viral genome in the producer cell line, but results in “self-inactivation” of the lentivirus after transduction of the target cel  (Yee et al., 1987; Yu et al., 1986; Zufferey et al., 1998). Once integrated into the transduced target cell, the lentiviral genome is no longer capable of producing packageable viral genome.
  • The number of genes from HIV-1 that are used in the system has been reduced to three (i.e. gag, pol, and rev).
  • The VSV-G gene from Vesicular Stomatitis Virus is used in place of the HIV-1 envelope (Burns et al., 1993; Emi e  al., 1991; Yee et al., 1994).
  • Genes encoding the structural and other components required for packaging the viral genome are separated onto four plasmids. All four plasmids have been engineered not to contain any regions of homology with each other to prevent undesirable recombination events that could lead to the generation of a replication-competent virus (Dull et al., 1998).
  • Although the three packaging plasmids allow expression in trans of proteins required to produce viral progeny (e.g. gal, pol, rev, env) in the 293FT producer cell line, none of them contain LTRs or the Ψ packaging sequence. This means that none of the HIV-1 structural genes are actually present in the packaged viral genome, and thus, are never expressed in the transduced target cell. No new replication-competent virus can be produced.
  • The lentiviral particles produced in this system are replication-incompetent and only carry the gene of interest. N  other viral species are produced.
  • Expression of the gag and pol genes from pLP1 has been rendered Rev-dependent by virtue of the HIV-1 RRE in the gag/pol mRNA transcript. Addition of the RRE prevents gag and pol expression in the absence of Rev (Dull et al., 1998).
  • A constitutive promoter (RSV promoter) has been placed upstream of the 5′ LTR in the pLenti expression vector to offset the requirement for Tat in the efficient production of viral RNA (Dull et al., 1998).

Biosafety Level 2

Despite the inclusion of the safety features discussed on the previous page, the lentivirus produced with this System can still pose some biohazardous risk since it can transduce primary human cells. For this reason, we highly recommend that you treat lentiviral stocks generated using this System as Biosafety Level 2 (BL-2) organisms and strictly follow all published BL-2 guidelines with proper waste decontamination. Furthermore, exercise extra caution when creating lentivirus carrying potential harmful or toxic genes (e.g. activated oncogenes).
For more information about the BL-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). This document may be downloaded at the following address: http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm

Important:  Handle all lentiviruses in compliance with established institutional guidelines. Since safety requirements for use and handling of lentiviruses may vary at individual institutions, we recommend consulting the health and safety guidelines and/or officers at your institution prior to use of the ViraPower™ Lentiviral Expression System.

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Methods

General Information

Introduction The ViraPower™ Lentiviral Expression System is designed to help you create a lentivirus to deliver and express a gene of interest in mammalian cells. Although the system has been designed to help you express your recombinant protein of interest in the simplest, most direct fashion, use of the system is geared towards those users who are familiar with the principles of retrovirus biology and retroviral vectors. We highly recommend that users possess a working knowledge of virus production and tissue 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) and Luciw (1996)
  • Retroviral and lentiviral vectors: see Naldini (1999), Naldini (1998), Yee (1999) and Pandya et al., (2001)


Generating Your pLenti Expression Construct

To generate a pLenti expression construct containing your gene of interest, refer to the manual for the vector you are using for instructions. Once you have created your expression construct, you will isolate plasmid DNA for transfection. Important: You should verify that your lentiviral plasmid has not undergone aberrant recombination b  performing an appropriate restriction enzyme digest. See the vector manual for details.

DNA Isolation Guidelines

Plasmid DNA for transfection into eukaryotic cells must be very clean and free from contamination with phenol and sodium chloride. Contaminants will kill the cells, and salt will interfere with lipid complexing, decreasing transfection efficiency.

When performing plasmid DNA isolation with commercially available kits from E. coli strains (such as Stbl3™) that are wild type for endonuclease 1 (endA1+), ensure that Solution I of the Lysis or Resuspension Buffer contains 10 mM EDTA. EDTA will inactivate the endonuclease and avoid DNA nicking and vector degradation. Alternatively, follow the instructions included the plasmid purification kits for endA1+ E. coli strains.

Do not use mini-prep plasmid DNA for lentivirus production. We recommend preparing lentiviral plasmid DNA using the S.N.A.P.™ MidiPrep Kit, which contains 10 mM EDTA in the Resuspension Buffer (see ordering information).

ViraPower™ Packaging Mix

The pLP1, pLP2, pLP/VSVG plasmids are provided in an optimized mixture to facilitate viral packaging of your pLenti expression vector following cotransfection into 293FT producer cells. The amount of the packaging mix (195 μg supplied as 1 μg/μl in TE Buffer, pH 8.0) and Lipofectamine™ 2000 transfection reagent (0.75 ml) supplied in the ViraPower™ Lentiviral Expression kit is sufficient to perform 20 cotransfections in 10 cm plates.

Note:
ViraPower™ Packaging Mix is available separately from Invitrogen or as part of the ViraPower™ Lentiviral Support Kits
.
293FT Cell Line

The human 293FT Cell Line is supplied with the ViraPower™ Lentiviral Expression kits to facilitate optimal lentivirus production (Naldini et al., 1996). The 293FT Cell Line, a derivative of the 293F Cell Line, stably and constitutively expresses the SV40 large T antigen from pCMVSPORT6TAg.neo and must be maintained in medium containing Geneticin®. For more information about pCMVSPORT6TAg.neo and how to culture and maintain 293FT cells, refer to the 293FT Cell Line manual. This manual is supplied with the ViraPower™ Lentiviral Expression kits, and is also available for downloading from our web site at www.invitrogen.com or by contacting Technical Support.

Note: The 293FT Cell Line is also available separately from Invitrogen.

The health of your 293FT cells at the time of transfection has a critical effect on the success of lentivirus production. Use of “unhealthy” cells will negatively affect the transfection efficiency, resulting in production of a low titer lentiviral stock. For optimal lentivirus production (i.e. producing lentiviral stocks with the expected titers), follow the guidelines below to culture 293FT cells before use in transfection:

  • Make sure that cells are healthy and greater than 90% viable.
  • Subculture and maintain cells in complete medium containing 0.1 mM MEM Non-Essential Amino Acids, 4 mM L-Glutamine, 1 mM sodium pyruvate, 500 μg/ml Geneticin® and 10% fetal bovine serum that is not heat-inactivated
  • Do not allow cells to overgrow before passaging.
  • Use cells that have been subcultured for less than 16 passages.


Positive Control

We recommend including a positive control vector in your cotransfection experiment to generate a control lentiviral stock that may be used to help you optimize expression conditions in your mammalian cell line of interest.

  • Each pLenti expression vector kit includes a positive control vector for use as an expression control (e.g. pLenti6/V5-GW/lacZ). For more information about the positive control vector supplied with each kit, refer to the appropriate expression vector manual.
  • A control lentiviral expression vector (pLenti6.2-GW/EmGFP) containing Emerald Green Fluorescent Protein (EmGFP) for fluorescent detection is available separately from Invitrogen.


Lipofectamine™ 2000


The Lipofectamine™ 2000 reagent supplied with the kit (Ciccarone et al., 1999) is a proprietary, cationic lipid-based formulation suitable for the transfection of nucleic acids into eukaryotic cells. Using Lipofectamine™ 2000 to transfect 293FT cells offers the following advantages:

  • Provides the highest transfection efficiency in 293FT cells
  • DNA-Lipofectamine™ 2000 complexes can be added directly to cells in culture medium in the presence of serum
  • Removal of complexes or medium change or addition following transfection are not required, although complexes can be removed after 4–6 hours without loss of activity


Note: 
Lipofectamine™ 2000 is available separately from Invitrogen or as part of the ViraPower™ Lentiviral Support Kits.


Opti-MEM®


To facilitate optimal formation of DNA-Lipofectamine™ 2000 complexes, we recommend using Opti-MEM® I Reduced Serum Medium available from Invitrogen.

Recommended Procedure

If you producing lentivirus for the first time using the ViraPower™ System and 293FT cells, you should perform the Forward Transfection procedure. This procedure requires plating the 293FT cells the day before transfection to obtain cells that are 90–95% confluent

Note:
In previous ViraPower™ manuals, this protocol was called the Alternate Transfection Method.

Alternate Transfection Method.

If you are an experienced lentivirus user and are familiar with the growth characteristics of 293FT cells, you may choose to perform the Reverse Transfection procedure. In this procedure, 293FT cells are added to media containing the DNA-Lipofectamine™ 2000 complexes.

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Producing Lentivirus in 293FT Cells

Introduction

Before you can create a stably transduced cell line expressing your gene of interest, you will first need to produce a lentiviral stock (containing the packaged pLenti expression construct) by cotransfecting the optimized packaging plasmid mix and your pLenti expression construct into the 293FT Cell Line. The following section provides protocols and instructions to generate a lentiviral stock.

Recommended Transfection Conditions

We produce lentiviral stocks in 293FT cells using the following optimized transfection conditions below. The amount of lentivirus produced using these recommended conditions (10 ml of virus at a titer of at least 1 × 105 transducing units (TU)/ml) is generally sufficient to transduce at least 1 × 106 cells at a multiplicity of infection (MOI) = 1. For example, 10 wells of cells plated at 1 × 105 cells/well in 6-well plates could each be transduced with 1 ml of a 1 × 105 TU/ml virus stock to achieve an MOI of 1.

Condition Quantity
Tissue culture plate size10 cm (one per lentiviral construct)
Number of 293FT cells to transfect6 × 106 cells
Amount of ViraPower™ Packaging Mix9 μg
Amount of pLenti expression plasmid3 μg
Amount of Lipofectamine™ 200036 μl

Note:   You may produce lentiviral stocks using other tissue culture formats, but keep in mind that optimization will be necessary to obtain the expected titers.

Materials Needed

You will need the following items:

  • ViraPower™ Packaging Mix (supplied in solution with the kit)
  • pLenti expression vector containing your gene of interest (0.1–3.0 μg/μl)
  • pLenti control vector containing lacZ (supplied with the kit) or EmGFP (available separately)
  • 293FT cells cultured in the appropriate medium (i.e. D-MEM containing 10% FBS, 4 mM L-Glutamine, 1 mM MEM Sodium Pyruvate, 0.1 mM MEM Non-Essential Amino Acids, and 1% penicillin-streptomycin, and 500 μg/ml Geneticin®) Note: MEM Sodium Pyruvate provides an extra energy source for the cells and is available from Invitrogen as a 100 mM stock solution (Catalog no. 11360-070).
  • Lipofectamine™ 2000 transfection reagent (supplied with the kit; store at 4°C and mix gently before use)
  • Opti-MEM® I Reduced Serum Medium (pre-warmed to 37°C)
  • Fetal bovine serum (FBS, Cat. no 16000-044)
  • Complete growth medium without antibiotics (i.e. D-MEM containing 10% FBS, 4 mM L-Glutamine, 0.1 mM MEM Non-Essential Amino Acids, and 1 mM MEM Sodium Pyruvate), pre-warmed to 37°C
  • Sterile, 10 cm tissue culture plates (one each for the lentiviral construct, positive control, and negative control)
  • Sterile, tissue culture supplies
  • 15 ml sterile, capped, conical tubes
  • Optional: Millex-HV 0.45 μm PVDF filters (Millipore, cat. no. SLHVR25LS) or equivalent
  • Cryovials


Forward Transfection Procedure


If you are a first time user, follow the procedure below to cotransfect 293FT cells. We recommend including a negative control (no DNA, no Lipofectamine™ 2000) in your experiment to help you evaluate your results.
  1. The day before transfection (Day 1), plate 293FT cells in a 10 cm tissue culture plate so that they will be 90–95% confluent on the day of transfection (i.e. 5 × 106 cells in 10 ml of growth medium containing serum). Do not include antibiotics in the medium.

  2. On the day of transfection (Day 2), remove the culture medium from the 293FT cells and replace with 5 ml of growth medium (or Opti-MEM® I Medium) containing serum. Do not include antibiotics in the medium.

  3. For each transfection sample, prepare DNA-Lipofectamine™ 2000 complexes as follows:

    • In a sterile 5 ml tube, dilute 9 μg of the ViraPower™ Packaging Mix and 3 μg of pLenti expression plasmid DNA (12 μg total) in 1.5 ml of Opti-MEM® I Medium without serum. Mix gently.
    • In a separate sterile 5 ml tube, mix Lipofectamine™ 2000 gently before use, then dilute 36 μl in 1.5 ml of Opti-MEM® I Medium without serum. Mix gently and incubate for 5 minutes at room temperature.
    • After the 5 minute incubation, combine the diluted DNA with the diluted Lipofectamine™ 2000. Mix gently.
    • Incubate for 20 minutes at room temperature to allow the DNALipofectamine ™ 2000 complexes to form. The solution may appear cloudy, but this will not impede the transfection.

  4. Add the DNA-Lipofectamine™ 2000 complexes dropwise to each plate of cells. Mix gently by rocking the plate back and forth. Incubate the cells overnight at 37°C in a humidified 5% CO2 incubator.

  5. The next day (Day 3), remove the medium containing the DNALipofectamine ™ 2000 complexes and replace with 10 ml complete culture medium without antibiotics. Incubate at 37°C in a humidified 5% CO2
    incubator. Note: Expression of the VSV G glycoprotein causes 293FT cells to fuse, resulting in the appearance of large, multinucleated cells known as syncytia. This morphological change is normal and does not affect production of the lentivirus.

  6. Harvest virus-containing supernatants 48–72 hours posttransfection (Day 4–5) by removing medium into to a 15 ml sterile, capped, conical tube. Minimal differences in viral yield are observed whether supernatants are collected at either 48 or 72 hours posttransfection. Caution: Remember that you are working with infectious virus at this stage. Follow the recommended guidelines for working with BL-2 organisms.

  7. Centrifuge supernatants at 3000 rpm for 15 minutes at 4°C to pellet debris.

  8. Optional: Filter the viral supernatants through a Millex-HV 0.45 μm or equivalent PVDF filter.

  9. Pipet viral supernatants into cryovials in 1 ml aliquots. Store viral stocks at –80°C. Proceed to Titering Your Lentiviral Stock.

Reverse Transfection Procedure

If you are an experienced user, you may use the rapid procedure below to cotransfect 293FT cells. We recommend including a negative control (no DNA, no Lipofectamine™ 2000) in your experiment to help you evaluate your results. You will need 6 × 106 293FT cells for each sample.

  1. On Day 1, prepare DNA-Lipofectamine™ 2000 complexes for each transfection sample as follows:

    • In a sterile 5 ml tube, dilute 9 μg of the ViraPower™ Packaging Mix and 3 μg of pLenti expression plasmid DNA (12 μg total) in 1.5 ml of Opti-MEM® I Medium without serum. Mix gently.
    • In a separate sterile 5 ml tube, mix Lipofectamine™ 2000 gently before use, then dilute 36 μl in 1.5 ml of Opti-MEM® I Medium without serum. Mix gently and incubate for 5 minutes at room temperature.
    • After the 5 minute incubation, combine the diluted DNA with the diluted Lipofectamine™ 2000. Mix gently.
    • Incubate for 20 minutes at room temperature to allow the DNALipofectamine ™ 2000 complexes to form. The solution may appear cloudy, but this will not impede the transfection.

  2. While DNA-lipid complexes are forming, trypsinize and count the 293FT cells. Resuspend the cells at a density of 1.2 × 106 cells/ml in growth medium (or Opti-MEM® I Medium) containing serum. Do not include antibiotics in the medium.

  3. Add the DNA-Lipofectamine™ 2000 complexes to a 10 cm tissue culture plate containing 5 ml of growth medium (or Opti-MEM® I Medium) containing serum. Do not include antibiotics in the medium.

  4. Add 5 ml of the 293FT cell suspension (6 × 106 total cells) to the plate containing media and DNA-Lipofectamine™ 2000 complexes. Mix gently by rocking the plate back and forth. Incubate cells overnight at 37°C in a humidified 5% CO2 incubator.

  5. The next day (Day 2), remove the medium containing the DNALipofectamine ™ 2000 complexes and replace with 10 ml complete culture medium without antibiotics. Incubate cells overnight at 37°C in a humidified 5% CO2 incubator.
    Note: Expression of the VSV G glycoprotein causes 293FT cells to fuse, resulting in the appearance of large, multinucleated cells known as syncytia. This morphological change is normal and does not affect production of the lentivirus.

  6. Harvest virus-containing supernatants 48–72 hours posttransfection (Day 3–4) by removing medium into a 15 ml sterile, capped, conical tube. Minimal differences in viral yield are observed whether supernatants are collected 48 or 72 hours posttransfection. Caution: Remember that you are working with infectious virus at this stage. Follow the recommended guidelines for working with BL-2 organisms.

  7. Centrifuge supernatants at 3000 rpm for 15 minutes at 4°C to pellet debris.

  8. Optional: Filter the viral supernatants through a Millex-HV 0.45 μm or equivalent PVDF filter.

  9. Pipet viral supernatants into cryovials in 1 ml aliquots. Store viral stocks at –80°C. Proceed to Titering Your Lentiviral Stock.


If you plan to use your lentiviral construct for in vivo applications, we recommend filtering your viral supernatant through a sterile, 0.45 μm low protein binding filter after the low-speed centrifugation step (above) to remove any remaining cellular debris. We recommend using Millex-HV 0.45 μm PVDF filters (Millipore, Catalog no. SLHVR25LS) for filtration. If you wish to concentrate your viral stock to obtain a higher titer, perform the filtration step first before concentrating your viral stock.

Concentrating Virus

It is possible to concentrate VSV-G pseudotyped lentiviruses using a variety of methods without significantly affecting their ability to transduce cells. If your cell transduction experiment requires that you use a relatively high MOI, you may wish to concentrate your virus before titering and proceeding to transduction. For details and guidelines to concentrate your virus supernatant by ultracentrifugation, refer to published reference sources (Yee, 1999).

Long-Term Storage

Store viral stocks at –80°C in cryovials for long-term storage. Repeated freezing and thawing is not recommended as it may result in loss of viral titer. When stored properly, viral stocks of an appropriate titer should be suitable for use for up to one year. After long-term storage, we recommend retitering your viral stocks before transducing your mammalian cell line of interest.

Scaling Up Virus Production

It is possible to scale up the cotransfection experiment to produce a larger volume of lentivirus, if desired. For example, we have scaled up the cotransfection experiment from a 10 cm plate to a T-175 cm2 flask and harvested up to 30 ml of viral supernatant. If you wish to scale up your cotransfection, remember that you will need to increase the number of cells plated and the amounts of DNA, Lipofectamine™ 2000, and medium used in proportion to the difference in surface area of the culture vessel.
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Titering Your Lentiviral Stock

Introduction

Before proceeding to transduction and expression experiments, we highly recommend determining the titer of your lentiviral stock. While this procedure is not required for some applications, it is necessary if:

  • You wish to control the number of integrated copies of the lentivirus
  • You wish to generate reproducible expression results


Guidelines and protocols are provided in this section to titer your lentiviral stock. Note: If you are using pLenti6.2-GW/EmGFP Expression Control Vector to produce a lentiviral stock, refer to the user manual for titer methods using fluorescent detection.

Caution

Remember that you will be working with media containing infectious virus. Follow the recommended Federal and institutional guidelines for working with BL-2 organisms.

  • Perform all manipulations within a certified biosafety cabinet.

  • Treat media containing virus with bleach.

  • Treat used pipettes, pipette tips, and other tissue culture supplies with bleach and dispose of as biohazardous waste.

  • Wear gloves, a laboratory coat, and safety glasses or goggles when handling viral stocks and media containing virus.


Experimental Outline

To determine the titer of lentiviral stocks, you will:

  1. Prepare 10-fold serial dilutions of your lentiviral stocks.

  2. Transduce the different dilutions of lentivirus in the presence of the polycation Polybrene® into a mammalian cell line (HT1080 is recommended).

  3. Select for stably transduced cells using Blasticidin.

  4. Stain and count the number of Blasticidin-resistant colonies in each dilution.


Factors Affecting Viral Titer

A number of factors can influence viral titers including:

  • The size of your gene of interest. Titers will decrease as the size of the insert increases. We have determined that virus titer drops approximately 2-fold for each kb over 4 kb of insert size. If you wish to produce lentivirus with an insert of >4 kb, you will need to concentrate the virus to obtain a suitable titer. The size of the wild-type HIV genome is approximately 10 kb. Since the size of the elements required for expression from pLenti vectors total approximately 4-4.4 kb, the size of your insert should not exceed 5.6 kb.

  • The characteristics of the cell line used for titering- We strongly recommend the human fibrosarcoma line HT1080 as the “gold standard” for reproducibly titering lentivirus. However, other cell lines may be used. In general, these cells should be an adherent, non-migratory cell line, and exhibit a doubling time in the range of 18-25 hours.

  • The age of your lentiviral stock- Viral titers may decrease with long-term (>1 year) storage at –80°C. If your lentiviral stock has been stored for longer than 6 months, we recommend titering your lentiviral stock prior to use.

  • Number of freeze/thaw cycles- Viral titers can decrease as much as 10% with each freeze/thaw cycle.

  • Improper storage of your lentiviral stock- Lentiviral stocks should be stored at –80°C in cryovials.


Selecting a Cell Line for Titering

We strongly recommend the human fibrosarcoma line HT1080 (ATCC, cat no. CCL-121) as the “gold standard” of  reproducibly titering lentivirus. However, you may wish to use the same mammalian cell line to titer your lentiviral stocks as you will use to perform your expression studies (e.g. if you are performing expression studies in a dividing cell line or a non-primary cell line). If you have more than one lentiviral construct, we recommend that you titer all of the lentiviral constructs using the same mammalian cell line. For more information on cells for titering, see Factors Affecting Viral Titer.

Antibiotic Selection

The pLenti expression constructs contain either the Blasticidin resistance gene (bsd) (Kimura et al., 1994) or the Zeocin™ resistance gene (Calmels et al., 1991; Drocourt et al., 1990) to allow for Blasticidin selection (Takeuchi et al., 1958; Yamaguchi et al., 1965) or Zeocin™ selection (Mulsant et al., 1988), respectively of mammalian cells that have stably transduced the lentiviral construct. If you have purchased a ViraPower™ Lentiviral Expression Kit, either Blasticidin or Zeocin™ is supplied. Blasticidin and Zeocin™ are also available separately from Invitrogen or as part of the appropriate ViraPower™ Lentiviral Support Kit

Important:  Cell density can affect the efficiency of Zeocin™ selection. For the most efficient Zeocin™ selection, cells should not be greater than 50% confluent.

Determining Antibiotic Sensitivity

Since you will be selecting for stably transduced cells using Blasticidin or Zeocin™, you must first determine the minimum concentration of Blasticidin or Zeocin™ required to kill your untransduced mammalian cell line (i.e. perform a kill curve experiment). Typically, concentrations ranging from 2-10 μg/ml Blasticidin or 50–1000 μg/ml Zeocin™ are sufficient to kill most untransduced mammalian cell lines. We recommend that you test a range of concentrations (see protocol below) to ensure that you determine the minimum concentration necessary for your cell line.

  1. Plate cells at approximately 25% confluence. Prepare a set of 6–7 plates. Allow cells to adhere overnight.

  2. The next day, substitute culture medium with medium containing varying concentrations of Blasticidin or Zeocin™, as appropriate.

  3. Replenish the selective media every 3–4 days, and observe the percentage of surviving cells.

  4. Determine the appropriate concentration of Blasticidin or Zeocin™ that kills the cells within 10–14 days after addition of antibiotic.


Effect of Zeocin™ on Sensitive and Resistant Cells

Zeocin™’s method of killing is quite different from that of other common antibiotics such as Blasticidin or Geneticin®. Zeocin™-sensitive cells do not round up and detach from the plate, but may exhibit the following morphological changes:

  • Vast increase in size (similar to the effects of cytomegalovirus infecting permissive cells)

  • Abnormal cell shape

  • Presence of large empty vesicles in the cytoplasm (breakdown of the endoplasmic reticulum and Golgi apparatus or scaffolding proteins)

  • Breakdown of plasma and nuclear membrane (appearance of many holes in these membranes)


Eventually, these “cells” will completely break down and only “strings” of protein will remain. Zeocin™-resistant cells should continue to divide at regular intervals to form distinct colonies. There should not be any distinct morphological changes in Zeocin™-resistant cells when compared to non-selected cells.

Using Polybrene® During Transduction

Lentivirus transduction may be enhanced if cells are transduced in the presence of hexadimethrine bromide (Polybrene®). For best results, we recommend performing transduction in the presence of Polybrene®. Note however, that some cells are sensitive to Polybrene® (e.g. primary neurons). Before performing any transduction experiments, you may want to test your cell line for sensitivity to Polybrene® at a range of 0-10 μg/ml. If your cells are sensitive to Polybrene® (e.g. exhibit toxicity or phenotypic changes), do not add Polybrene® during transduction. In this case, cells should still be successfully transduced with your lentivirus.

Preparing and Storing Polybrene®

Follow the instructions below to prepare Polybrene® (Sigma, Catalog no. H9268):

  1. Prepare a 6 mg/ml stock solution in deionized, sterile water.

  2. Filter-sterilize and dispense 1 ml aliquots into sterile microcentrifuge tubes.

  3. The working stock may be stored at 4°C for up to 2 weeks. Store at –20°C for long-term storage (up to 1 year). Do not freeze/thaw the stock solution more than 3 times as this may result in loss of activity.


Materials Needed

You will need the following items:

  • Your pLenti lentiviral stock (store at –80°C until use)

  • Adherent mammalian cell line of choice

  • Complete culture medium for your cell line

  • 6 mg/ml Polybrene®, if desired

  • 6-well tissue culture plates

  • 10 cm tissue culture plates (for Zeocin™ selection only)

  • Blasticidin (10 mg/ml stock) or Zeocin™ (100 mg/ml stock), as appropriate for selection

  • Crystal violet (Sigma, Catalog no. C3886; prepare a 1% crystal violet solution in 10% ethanol)

  • Phosphate-Buffered Saline


Transduction and Titering Procedure

Follow the procedure below to determine the titer of your lentiviral stock using the mammalian cell line of choice. You will use at least one 6-well plate for every lentiviral stock to be titered (one mock well plus five dilutions). Note: If you have generated a lentiviral stock of a lacZ expression control (e.g. pLenti6/V5-GW/lacZ), we recommend titering this stock as well. If you are using pLenti6.2-GW/EmGFP, refer to the user manual for the vector for the titering protocol.

  1. The day before transduction (Day 1), trypsinize and count the cells, plating them in a 6-well plate such that they will be 30–50% confluent at the time of transduction. Incubate cells at 37°C overnight in a humidified 5% CO2 incubator. Example: When using HT1080 cells, we usually plate 2 × 105 cells per well in a 6-well plate.

  2. On the day of transduction (Day 2), thaw your lentiviral stock and prepare 10-fold serial dilutions ranging from 10-2 to 10-6. For each dilution, dilute the lentiviral stock into complete culture medium to a final volume of 1 ml. DO NOT vortex. Note: You may prepare a wider range of serial dilutions (10-2 to 10-8), if desired.

  3. Remove the culture medium from the cells. Mix each dilution gently by inversion and add to one well of cells (total volume = 1 ml).

  4. Add Polybrene® (if desired) to each well to a final concentration of 6 μg/ml. Swirl the plate gently to mix. Incubate at 37°C overnight in a humidified 5% CO2 incubator.

  5. The following day (Day 3), remove the media containing virus and replace with 2 ml of complete culture medium. Incubate at 37°C overnight in a humidified 5% CO2 incubator.

  6. The following day (Day 4), treat cells as follows:

    • For Blasticidin selection, remove the medium and replace with complete culture medium containing the appropriate amount of Blasticidin 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 culture 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 10-12 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. Add crystal violet solution (1 ml for 6-well dish; 5 ml for 10 cm plate) and incubate for 10 minutes at room temperature.

  10. Remove the crystal violet stain and wash the cells with PBS. Repeat wash.

  11. Count the blue-stained colonies and determine the titer of your lentiviral stock.


What You Should See

When titering pLenti lentiviral stocks using HT1080 cells, we generally obtain titers ranging from 1 - 5 × 105 (for unconcentrated virus) up to 2 × 107 (for concentrated virus) transducing units (TU)/ml.

Example of Expected Results

In this experiment, a Lenti6/V5-GW/lacZ lentiviral stock was generated using the protocol above and was concentrated by ultracentrifugation. HT1080 cells were transduced with 10-fold serial dilutions of the lentiviral supernatant (102 to 106 dilutions) or untransduced (mock) following the protocol. At 48 hours post-transduction, the cells were placed under Blasticidin selection (10 μg/ml). After 10 days of selection, the cells were stained with crystal violet and colonies were counted.

Next Steps

It is important to note that user experience, the nature of the gene, and vector backbone may affect virus titer. If the titer of your unconcentrated virus is suitable (i.e. 1 × 105 TU/ml or higher), proceed to Transduction of Cells With Lentivirus. If the titer of your concentrated lentiviral stock is less than 1 × 105 TU/ml, we recommend producing a new lentiviral stock. See the Troubleshooting section for more tips and guidelines to optimize your viral yield.

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Transduction and Analysis

Introduction

Once you have generated a lentiviral stock with a suitable titer, you are ready to transduce the lentiviral construct into the mammalian cell line of choice and assay for expression of your recombinant protein. Guidelines are provided below.

Your lentiviral construct contains a deletion in the 3′ LTR that leads to self-inactivation of the lentivirus after transduction into mammalian cells. Once integrated into the genome, the lentivirus can no longer produce  packageable virus.

Transient vs. Stable Expression

After transducing your lentiviral construct into the mammalian cell line of choice, you may assay for expression of your gene of interest in the following ways:

  • Pool a heterogeneous population of cells and test for expression directly after transduction (i.e. “transient” expression). Note that you must wait for a minimum of 48–72 hours after transduction before harvesting your cells to allow expressed protein to accumulate in transduced cells.
  • Select for stably transduced cells using Blasticidin or Zeocin™, as appropriate. This requires a minimum of 10-12 days after transduction, but allows generation of clonal cell lines that stably express the gene of interest. Note: We have observed stable expression of a target gene for at least 6 weeks following transduction and selection.


Determining Antibiotic Sensitivity for Your Cell Line

If you wish to select for stably transduced cells, you must first determine the minimum concentration of Blasticidin or Zeocin™, as appropriate, required to kill your untransduced mammalian cell line (i.e. perform a kill curve experiment). If you titered your lentiviral construct in the same mammalian cell line that you are using to perform your stable expression experiment, then you may use the same concentration of Blasticidin or Zeocin™ for selection that you used for titering.

Multiplicity of Infection (MOI)

To obtain optimal expression of your gene of interest, you will need to transduce the lentiviral construct into your mammalian cell line of choice using a suitable MOI. MOI is defined as the number of virus particles per cell and generally correlates with the number of integration events and as a result, expression of your gene of interest. Typically, expression levels increase linearly as the MOI increases.

Determining the Optimal MOI

A number of factors can influence optimal MOI including the nature of your mammalian cell line (e.g. non-dividing vs. dividing cell type; see Recommendation below), its transduction efficiency, your application of interest, and the nature of your gene of interest. If you are transducing your lentiviral construct into the mammalian cell line of choice for the first time, we recommend using a range of MOI (e.g. 0, 0.5, 1, 2, 5, 10) to determine the MOI required to obtain the optimal expression of your protein for your application.

In general, we have found that 80-90% of the cells in an actively dividing cell line (e.g. HT1080) express a target gene when transduced at an MOI of ~1. Some nondividing cell types transduce lentiviral constructs less efficiently. For example, only about 50% of the cells in a culture of primary human fibroblasts express a target gene when transduced at an MOI of ~1. If you are transducing your lentiviral construct into a non-dividing cell type, you may need to increase the MOI (e.g. MOI = 10) to achieve optimal expression levels for your recombinant protein.

Positive Control

Control lentiviral vectors expressing lacZ or EmGFP are available for optimization. If you have generated a lentiviral stock of a lacZ expression control (e.g. pLenti6/V5-GW/lacZ), we recommend using the stock to help you determine the optimal MOI for your particular cell line and application. Once you have transduced the control lentivirus into your mammalian cell line of choice, the gene encoding β-galactosidase or EmGFP will be constitutively expressed and can be easily assayed (refer to the expression vector or expression control vector manual for assay methods).

Viral supernatants are generated by harvesting spent media containing virus from the 293FT producer cells. Spent media lacks nutrients and may contain some toxic metabolic waste products. If you are using a large volume of viral supernatant to transduce your mammalian cell line (e.g. 1 ml of viral supernatant per well in a 6-well plate), note that growth characteristics or morphology of the cells may be affected during transduction. These effects are generally alleviated after transduction when the media is replaced with fresh, complete media.


Materials Needed


You will need the following items:

  • Your titered lentiviral stock (store at –80°C until use)
  • Mammalian cell line of choice
  • Complete culture medium for your cell line
  • 6 mg/ml Polybrene®, if desired
  • Appropriately sized tissue culture plates for your application
  • Blasticidin or Zeocin™, as appropriate (if selecting for stably transduced cells)



Transduction Procedure

Follow the procedure below to transduce the mammalian cell line of choice with your lentiviral construct. Reminder: If you are performing Zeocin™ selection, remember that cells should not be confluent at the time of selection (see Step 6 below). Plate your cells accordingly.

  1. Plate cells in complete media as appropriate for your application.

  2. On the day of transduction (Day 1), thaw your lentiviral stock, and if necessary, dilute the appropriate amount of virus into fresh complete medium to obtain a suitable MOI. Keep the total volume of medium containing virus as low as possible to maximize transduction efficiency. DO NOT vortex.

  3. Remove the culture medium from the cells. Mix the medium containing virus gently by pipetting and add to the cells.

  4. Add Polybrene® (if desired) to a final concentration up to 10 μg/ml. Swirl the plate gently to mix. Incubate at 37°  in a humidified 5% CO2 incubator overnight. Note: If you are transducing cells with undiluted viral stock and are concerned about possible toxicity or growth effects caused by overnight incubation, it is possible to incubate cells for as little as 6 hours prior to changing medium.

  5. The following day (Day 2), remove the medium containing virus and replace with fresh, complete culture medium. Incubate at 37°C in a humidified 5% CO2 incubator overnight.

  6. The following day (Day 3), perform one of the following:

    • Harvest the cells and assay for expression of your recombinant protein if you are performing transient expression experiments.

    • Remove the medium and replace with fresh, complete medium containing the appropriate amount of Blasticidin or Zeocin™, as appropriate to select for stably transduced cells. Proceed to Step 7.

  7. Replace medium with fresh medium containing antibiotic every 3-4 days until antibiotic-resistant colonies can be identified (generally 10–12 days after selection).

  8. Pick at least 5 antibiotic-resistant colonies (see Note below) and expand each clone to assay for expression of the recombinant protein.



Integration of the lentivirus into the genome is random. Depending upon the influence of the surrounding genomic sequences at the integration site, you may see varying levels of recombinant protein expression from different antibiotic-resistant clones. We recommend testing at least 5 antibiotic-resistant clones and selecting the clone that provides the optimal expression of your recombinant protein for further studies.

Detecting Recombinant Protein

You may use any method of choice to detect your recombinant protein of interest including functional analysis, immunofluorescence, or western blot. If you have cloned your gene of interest in frame with an epitope tag, you may easily detect your recombinant protein in a western blot using an antibody to the epitope tag

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Troubleshooting

The table below lists some potential problems and possible solutions that may help you troubleshoot your cotransfection and titering experiments.

Generating the Lentiviral Stock

Problem   
Cause
Solution
Low viral titerLow transfection efficiency:

  • Used poor quality expression construct plasmid DNA (i.e. plasmid DNA from a mini-prep)
  • Unhealthy 293FT cells; cells exhibit low viability
  • Cells transfected in media containing antibiotics (i.e.
    Geneticin®)
  • Plasmid DNA:transfection reagent ratio incorrect
  • Insufficient co-transfection
  • 293FT cells plated too sparsely

 

  • Do not use mini-prep plasmid DNA for transfection. Use the S.N.A.P.™ Midiprep DNA Isolation Kit or CsCl gradient centrifugation to prepare plasmid DNA.
  • Use healthy 293FT cells under passage 16; do not overgrow.
  • Although Geneticin® is required for stable maintenance of 293FT cells, Do not add Geneticin® to media during transfection as this reduces transfection efficiency and causes cell death.
  • Use a DNA (in μg):Lipofectamine™ 2000 (in μl) ratio ranging from 1:2 to 1:3.
  • Use more DNA/ Lipofectamine™ 2000 (keeping the ratios the same). For example, use 5 μg of lentiviral vector, 15 μg of packaging mix, and 60 μl of Lipofectamine™ 2000 for transfection.
  • Plate cells such that they are 90–95% confluent at the time of transfection OR use the Reverse Transfection protocol
 Transfected cells not cultured in
media containing sodium
pyruvate
One day after transfection, remove media containing DNA-lipid complexes and replace with media containing sodium pyruvate.
Sodium pyruvate provides an extra energy source for the cells.
 Viral supernatant harvested too
early
Viral supernatants can generally be collected 48–72 hours posttransfection. If many cells are still attached to the plate and look healthy at this point, wait an additional 24 hours before harvesting the viral supernatant.

Viral supernatant too diluteConcentrate your virus (Yee, 1999).

 Viral supernatant frozen and
thawed multiple times
Do not freeze/thaw viral supernatant more than 3 times.

Poor choice of titering cell line
Use HT1080 cells or another adherent cell line

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Generating the Lentiviral Stock, continued

Problem   
Cause
Solution
Low viral titer, continued
Gene of interest is toxic to cells
Do not generate constructs containing
activated oncogenes or harmful genes.
 Gene of interest is largeViral titers generally decrease as the size of the insert increases. Concentrate the virus if titer is low  Inserts larger than 5.6 kb are not recommended.
 Polybrene® not included during transductionTransduce the lentiviral construct into cells in
the presence of Polybrene®.

Lipofectamine™ 2000 handled incorrectly
  • Store at 4°C. Do not freeze.
  • Mix gently by inversion. Do not vortex.

No colonies obtained
upon titering
Too much antibiotic used for selectionDetermine the antibiotic sensitivity of your cell
line by performing a kill curve experiment,
and use the minimum concentration required
to kill your untransduced cell line.

Viral stocks stored incorrectly
Aliquot and store stocks at –80°C. Do not
freeze/thaw more than 3 times.

Polybrene® not included during
transduction
Transduce the lentiviral construct into cells in
the presence of Polybrene®.
Titer indeterminable;
cells confluent

Too little antibiotic used for selection
Increase amount of antibiotic.

Zeocin™ selection performed on confluent cells
Before adding selective medium, trypsinize
transduced cells and replate in a larger tissue
culture plate.

Viral supernatant not diluted sufficiently
Titer lentivirus using a wider range of 10-fold
serial dilutions (e.g. 10-2 to 10-8).

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The table below lists some potential problems and possible solutions that may help you troubleshoot your transduction and expression experiment.

Transducing Mammalian Cells

Problem    Cause Solution
No expression of the gene of interest
Promoter silencing
  • Lentiviral constructs may integrate into a chromosomal region that silences the CMV promoter. Screen multiple antibiotic-resistant clones and select the one with the highest expression levels.
  • Use pLenti6/UbC/V5-DEST to generate your lentiviral construct, containing a cellular promoter that is not subject to silencing
 Viral stocks stored incorrectlyAliquot and store stocks at –80°C. Do not
freeze/thaw more than 3 times.
   
Poor expression of the gene of interestLow transduction efficiency:
  • Polybrene® not included during transduction
  • Non-dividing cell type used
  • Transduce the lentiviral construct into cells in the presence of Polybrene®.
  • Transduce your lentiviral construct into cells using a higher MOI.

MOI too lowTransduce your lentiviral construct into cells
using a higher MOI.

Too much antibiotic used for selectionDetermine the antibiotic sensitivity of your cell
line by performing a kill curve. Use the minimum antibiotic concentration required to kill your untransduced cell line.

Cells harvested too soon after transductionDo not harvest cells until at least 48–72 hours
after transduction to allow expressed protein
to accumulate in transduced cells.
 Gene of interest is toxic to cellsGenerating constructs containing activated
oncogenes or potentially harmful genes is not
recommended.
Cytotoxic effects
observed after
transduction
Large volume of viral
supernatant used for
transduction
  • Remove the “spent” media containing virus and replace with fresh, complete media.
  • Concentrate the virus (Yee, 1999).
 Polybrene® used during transductionVerify the sensitivity of your cells to Polybrene®. If cells are sensitive, omit the Polybrene® during transduction.
 Too much antibiotic used for selectionDetermine the antibiotic sensitivity of your
cell line by performing a kill curve. Use the
minimum concentration of antibiotic required
to kill your untransduced cell line.
 Gene of interest is toxic to cellsTry a different cell line.

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References

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