ViraPower Lentiviral Expression Systems—for high-level expression in dividing and non-dividing mammalian cells

The Invitrogen 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 Invitrogen 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 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 Invitrogen 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).

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 Invitrogen 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 Invitrogen 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  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 Invitrogen DNA-Lipofectamine 2000 complexes, we recommend using Invitrogen 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.

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.

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.

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 Invitrogen 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.

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

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

Generating the Lentiviral Stock

Generating the Lentiviral Stock, continued

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

Transducing Mammalian Cells

1. Andersson, S., Davis, D. L., Dahlbäck, H., Jörnvall, H., and Russell, D. W. (1989) Cloning, Structure, and Expression of the Mitochondrial Cytochrome P-450 Sterol 26-Hydroxylase, a Bile Acid Biosynthetic Enzyme. J. Biol. Chem. 264, 8222-8229
2. Boshart, M., Weber, F., Jahn, G., Dorsch-Häsler, K., Fleckenstein, B., and Schaffner, W. (1985) A Very Strong Enhancer is Located Upstream of an Immediate Early Gene of Human Cytomegalovirus. Cell 41, 521-530
3. Buchschacher, G. L., Jr., and Wong-Staal, F. (2000) Development of Lentiviral Vectors for Gene Therapy for Human Diseases. Blood 95, 2499-2504
4. Burns, J. C., Friedmann, T., Driever, W., Burrascano, M., and Yee, J.-K. (1993) Vesicular Stomatitis Virus G Glycoprotein Pseudotyped Retroviral Vectors: Concentration to a Very High Titer and Efficient Gene Transfer into Mammalian and Nonmammalian Cells. Proc. Natl. Acad. Sci. USA 90, 8033-8037
5. Calmels, T., Parriche, M., Burand, H., and Tiraby, G. (1991) High Efficiency Transformation of Tolypocladium geodes Conidiospores to Phleomycin Resistance. Curr. Genet. 20, 309-314
6. Ciccarone, V., Chu, Y., Schifferli, K., Pichet, J.-P., Hawley-Nelson, P., Evans, K., Roy, L., and Bennett, S. (1999) LipofectamineTM 2000 Reagent for Rapid, Efficient Transfection of Eukaryotic Cells. Focus 21, 54-55
7. Drocourt, D., Calmels, T. P. G., Reynes, J. P., Baron, M., and Tiraby, G. (1990) Cassettes of the Streptoalloteichus hindustanus ble Gene for Transformation of Lower and Higher Eukaryotes to Phleomycin Resistance. Nucleic Acids Res. 18, 4009
8. Dull, T., Zufferey, R., Kelly, M., Mandel, R. J., Nguyen, M., Trono, D., and Naldini, L. (1998) A Third- Generation Lentivirus Vector with a Conditional Packaging System. J. Virol. 72, 8463-8471
9. Emi, N., Friedmann, T., and Yee, J.-K. (1991) Pseudotype Formation of Murine Leukemia Virus with the G Protein of Vesicular Stomatitis Virus. J. Virol. 65, 1202-1207
10. Gorman, C. M., Merlino, G. T., Willingham, M. C., Pastan, I., and Howard, B. H. (1982) The Rous Sarcoma Virus Long Terminal Repeat is a Strong Promoter When Introduced into a Variety of Eukaryotic Cells by DNA-mediated Transfection. Proc. Natl. Acad. Sci. USA 79, 6777-6781
11. Izumi, M., Miyazawa, H., Kamakura, T., Yamaguchi, I., Endo, T., and Hanaoka, F. (1991) Blasticidin S-Resistance Gene (bsr): A Novel Selectable Marker for Mammalian Cells. Exp. Cell Res. 197, 229- 233
12. Kimura, M., Takatsuki, A., and Yamaguchi, I. (1994) Blasticidin S Deaminase Gene from Aspergillus terreus (BSD): A New Drug Resistance Gene for Transfection of Mammalian Cells. Biochim. Biophys. ACTA 1219, 653-659
13. Lewis, P. F., and Emerman, M. (1994) Passage Through Mitosis is Required for Oncoretroviruses but not for the Human Immunodeficiency Virus. J. Virol. 68, 510-516
14. Luciw, P. A. (1996) in Fields Virology (Fields, B. N.,Knipe, D. M., Howley, P. M., Chanock, R. M., Melnick, J. L., Monath, T. P., Roizman, B., and Straus, S. E., eds), 3rd Ed., pp. 1881-1975, Lippincott-Raven Publishers, Philadelphia, PA
15. Mulsant, P., Tiraby, G., Kallerhoff, J., and Perret, J. (1988) Phleomycin Resistance as a Dominant Selectable Marker in CHO Cells. Somat. Cell Mol. Genet. 14, 243-252
16. Naldini, L. (1998) Lentiviruses as Gene Transfer Agents for Delivery to Non-dividing Cells. Curr. Opin. Biotechnol. 9, 457-463
17. Naldini, L. (1999) in The Development of Human Gene Therapy (Friedmann, T., ed), pp. 47-60, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
18. Naldini, L., Blomer, U., Gage, F. H., Trono, D., and Verma, I. M. (1996) Efficient Transfer, Integration, and Sustained Long-Term Expression of the Transgene in Adult Rat Brains Injected with a Lentiviral Vector. Proc. Natl. Acad. Sci. USA 93, 11382-11388
19. Nelson, J. A., Reynolds-Kohler, C., and Smith, B. A. (1987) Negative and Positive Regulation by a Short Segment in the 5´-Flanking Region of the Human Cytomegalovirus Major Immediate-Early Gene. Molec. Cell. Biol. 7, 4125-4129
20. Pandya, S., Klimatcheva, E., and Planelles, V. (2001) Lentivirus and foamy virus vectors: novel gene therapy tools. Expert Opinion on Biological Therapy 1, 17-40
21. Takeuchi, S., Hirayama, K., Ueda, K., Sakai, H., and Yonehara, H. (1958) Blasticidin S, A New Antibiotic. The Journal of Antibiotics, Series A 11, 1-5
22. Yamaguchi, H., Yamamoto, C., and Tanaka, N. (1965) Inhibition of Protein Synthesis by Blasticidin S. I. Studies with Cell-free Systems from Bacterial and Mammalian Cells. J. Biochem (Tokyo) 57, 667- 677
23. Yee, J.-K., Miyanohara, A., LaPorte, P., Bouic, K., Burns, J. C., and Friedmann, T. (1994) A General Method for the Generation of High-Titer, Pantropic Retroviral Vectors: Highly Efficient Infection of Primary Hepatocytes. Proc. Natl. Acad. Sci. USA 91, 9564-9568
24. Yee, J. K. (1999) in The Development of Human Gene Therapy (Friedmann, T., ed), pp. 21-45, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
25. Yee, J. K., Moores, J. C., Jolly, D. J., Wolff, J. A., Respess, J. G., and Friedmann, T. (1987) Gene Expression from Transcriptionally Disabled Retroviral Vectors. Proc. Natl. Acad. Sci. USA 84, 5197-5201
26. Yu, S. F., Ruden, T. v., Kantoff, P. W., Garber, C., Seiberg, M., Ruther, U., Anderson, W. F., Wagner, E. F., and Gilboa, E. (1986) Self-Inactivating Retroviral Vectors Designed for Transfer of Whole Genes into Mammalian Cells. Proc. Natl. Acad. Sci. USA 83, 3194-3198
27. Zufferey, R., Dull, T., Mandel, R. J., Bukovsky, A., Quiroz, D., Naldini, L., and Trono, D. (1998) Self-inactivating Lentivirus Vector for Safe and Efficient in vivo Gene Delivery. J. Virol. 72, 9873-9880