The authors would like to acknowledge Michael Chou, Bingbing Dai, and Liang Xiao for contributing data for this manuscript. We also acknowledge Dr. John Gray for the generous gifts of reagents used in this study. P.B. is supported by a postdoctoral fellowship from the National Cancer Center. This research was supported by grants from the National Institute of Health (R01AI68978, P01CA132681 and RCA170820A), a grant from the Bill and Melinda Gates Foundation, a translational acceleration grant from the Joint Center for Translational Medicine and a grant from the California HIV/AIDS Research Program.

DC-LV stable producer cells are constructed based on the GPR packaging cell line 11 that contains the necessary lentiviral components gagpol, rev and the tet-off system. First, retroviral transduction is used to generate a GPRS packaging cell line that encodes a tet-dependent SVGmu glycoprotein. Then, concatemer array transfection is used to transfect the GPRS cell line with a lentiviral vector transgene such as GFP. This stable producer cell line, designated as LV-MGFP, can be tested in vitro and in vivo for its ability to produce a DC-LV vaccine against GFP.
1. Generating a Tet-dependent SVGmu Cell Line
Plasmid pRX-SVGmu is a construct in which DC-specific glycoprotein SVGmu is cloned downstream of the tTA-advanced promoter of retroviral plasmid pRetroX-Tet-off 1 (Figure 1C). Culture 293T cells in D10 (Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 2 mM L-glutamine). Culture GPR packaging cell line in D10 with doxycycline (1 ng/ml) and puromycin (2 &mu;g/ml). All cells are cultured in a humidified 37 &deg;C incubator with 5% CO2.
<ol>
	<li>16-18 hr before transient transfection, plate 2 x 106 293T cells in 4 ml of D10 in a 6-cm tissue culture dish such that the cells approach 90% confluency at the time of transfection.</li>
	<li>Transfection of retroviral plasmids: mix 100 &mu;l 1.25 M CaCl2 solution, sterile Milli-Q water and the following plasmids: 5 &mu;g pRX-SVGmu, 2.5 &mu;g pGag-Pol, 2.5 &mu;g pVSV-G. The final volume is 500 &mu;l. To a 5 ml polystyrene round-bottom tube, add 500 &mu;l 2X HBS (50 mM HEPES, 10 mM KCl, 12 mM Dextrose, 280 mM NaCl, 1.5 mM Na2HPO4*7H2O, pH 7.05). Add the plasmid mixture dropwise into the 2X HBS buffer while bubbling the buffer vigorously with a glass Pasteur pipette. After adding the mixture, continue bubbling for another 30 sec. Then, add the whole mixture onto the 293T cells in the 6-cm culture dish, and incubate at 37 &deg;C.</li>
	<li>4 hr after transfection, carefully replace the medium in the culture dish with 4 ml pre-heated D10.</li>
	<li>Spin infection: 48 hr after transfection, harvest SVGmu-encoding retroviral particles in the supernatant by passing the supernatant through a 0.45-&mu;m filter. Plate the GPR packaging cells in a 24-well dish at 2 x 104 cells/well. Add filtered supernatant to GPR packaging cells in the dish (2 ml/well) and centrifuge cells for 90 min at 1,050 x g and 25 &deg;C. Change medium into fresh D10 with doxycycline (1 ng/ml) after spin-infection (2 ml/well).</li>
	<li>72 hr post-transfection, expand the culture of transduced packaging cells in D10 with doxycycline (1 ng/ml) and puromycin (2 ng/ml).</li>
	<li>To confirm expression of SVGmu, culture the cells without doxycycline for 48 hr. Measure surface expression of SVGmu with flow cytometry using anti-Sindbis serum. These cells are designated as GPRS packaging cell line.</li>
</ol>
2. Construct DC-LV Producer Cells by Concatemer Array Transfection
Lentiviral transfer plasmid TL20-GFP is a self-inactivating lentiviral transfer vector plasmid based on pCL20c-MSCV-GFP with a Dox-regulatable viral RNA genome expression system and replacement of the cytomegalovirus (CMV) enhancer with 7 tet operators(Figure 1C) 11,12,15. Plasmid PGK-ble is a bleomycin resistant (ble) cassette driven by a weak PGK promoter 2.
<ol>
	<li>Digest 20 &mu;g plasmid TL20-GFP with the restriction enzyme SfiI at 50 &deg;C. Digest 20 &mu;g plasmid PGK-ble with PflMI at 37 &deg;C.</li>
	<li>Purify the DNA fragments (the PGK-ble cassette is 1011 bp and the vector TL20-GFP is 6861 bp) by agarose gel electrophoresis.</li>
	<li>Ligate the TL20-GFP vector and PGK-ble cassette at a molar ratio of 25:1 (NEB T4 DNA Ligase). Incubate overnight at room temperature.</li>
	<li>After overnight ligation, purify DNA from the ligation mixture (Qiagen DNeasy Blood and Tissue Kit). Ensure the amount of purified DNA is around 5 &mu;g.</li>
	<li>16-18 hr before transfection, plate GPRS packaging cells in a 6-cm tissue culture dish such that the confluency will be about 90% at the time of transfection.</li>
	<li>Use the calcium phosphate transfection method described in step 1.2 to transfect 5 &mu;g of the purified concatemeric DNA into the GPRS packaging cells in the 6-cm tissue culture dish. 4 hr after transfection, carefully remove the medium and replace with 4 ml pre-heated D10 with doxycycline (1 ng/ml).</li>
	<li>48 hr after transfection, change the medium into D10 with doxycycline (1 ng/ml) and puromycin (2 &mu;g/ml). Select for transfected clones by adding zeocin (50 &mu;g/ml) in the culture medium. Culture the cells for about 2 weeks until cell colonies can be seen at the bottom of the dishes.</li>
	<li>Label the cell colonies at the bottom of the dishes. Take 24-well tissue culture dishes, number the wells and add to each well 2 ml D10 containing zeocin (50 &mu;g/ml), doxycycline (1 ng/ml), and puromycin (2 &mu;g/ml). Aspirate the medium of the transduced cells, then add one drop of trypsin onto each of the colonies for less than one minute. Then, add one or more drops of D10 on the same colonies. Pick up colonies one by one with a pipette and transfer them into separate wells of the 24-well tissue culture plate.</li>
	<li>Culture and expand all cell clones in D10 containing zeocin (50 &mu;g/ml), doxycycline (1 ng/ml) and puromycin (2 &mu;g/ml) for the evaluation of viral producing ability.</li>
</ol>
3. Evaluate Viral Production of Each Cell Clone
<ol>
	<li>Trypsinize the producer cells and plate around 4x106 cells in 6-cm tissue culture dish in D10 without doxycycline such that the confluency exceeds 90%. Replace the medium with fresh pre-heated D10 daily and collect the medium for the titer assay 72 hr after removing dox.</li>
	<li>Harvest the viral supernatant by filtering the medium with a 0.45-&mu;m filter.</li>
	<li>Plate target cells expressing the DC-SIGN receptor (e.g. 293T.hDC-SIGN or mouse bone marrow derived dendritic cells 16) and 293T as a negative control in 96-well culture dishes, 1 x 104 cells / well. Add 2-fold serial dilutions of the viral supernatant to the wells (100 &mu;l/well). Usually, 6-8 dilutions are sufficient to reach the range in which transduced GFP-positive cells are in a linear relationship to the amount of virus added.</li>
	<li>Centrifuge cells and replace the medium as described in step 1.4. BMDCs should be cultured in RPMI medium with 10% FBS and GM-CSF (1:20 J558L conditioned medium).</li>
	<li>Measure the GFP expression in transduced cells by flow cytometry 4-6 days post-transduction. The lentiviral vector titer is calculated in the vector dilution range when the percentage of GFP-positive cells and vector amount are in a linear relationship. Choose the cell clone with the highest viral production for subsequent applications.</li>
</ol>
4. Produce and Concentrate Lentiviral Vectors
<ol>
	<li>Culture the producer cell line (LV-MGFP) in 15-cm tissue culture dishes.</li>
	<li>Trypsinize the producer cells and plate cells in 15-cm tissue culture dishes at greater than 90% confluency in fresh D10 without doxycycline. Change medium with fresh, pre-heated D10 daily.</li>
	<li>At the time of peak viral production (as determined by the user), harvest the viral supernatant and filter it using a 0.45-&mu;m filter. Load the filtered supernatant into thick wall 32.5 ml ultracentrifuge tubes, seal with parafilm and centrifuge at 50,000 x g, 4 &deg;C for 90 min. Thoroughly resuspend the pellet in 50 &mu;l or an appropriate volume of PBS or HBSS depending on the application.</li>
</ol>
5. Immunize Mice In vivo and Analyze Antigen-specific Immune Response
<ol>
	<li>Produce high-titer virus as described in section 4.</li>
	<li>Inject BALB/c mice (6-8 week old, female) via a footpad route with the vector suspension (25 &mu;l per footpad).</li>
	<li>2 weeks after immunization, isolate spleen and harvest splenocytes. Culture splenocytes with GFP epitope peptides and analyze the presence of GFP-specific CD8+ T cells using intracellular cytokine staining, as previously described 17 (Figure 3B).</li>
</ol>

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The authors declare that they have no competing financial interests.

Here, we have outlined a method for producing large quantities of lentiviral vectors using 293T cells stably transduced with all lentiviral components under the tet off regulatory system. To date, most protocols to produce lentiviral vectors rely on standard calcium phosphate transient transfection (see 18, for example). This approach has been successful on a clinical scale, but it may suffer from some limitations not likely to be present in scaling up production from stable cell lines. For example, co-transfe...

Many vector systems have been developed for gene delivery. Vectors based on lentiviruses have been among the most commonly studied viral system. These vectors are advantageous because they can efficiently transduce both dividing and non-dividing cells 1, achieve long-term expression due to integration into the host genome, exhibit low natural anti-vector immunity in most human populations 2, and have a low potential for genotoxicity from insertional mutagenesis 3,4.
Production of lentiviruses has always been colored by safety concerns. Lentiviral vectors are generally derived from HIV-1, the etiological agent of AIDS. Transient transfection of individual components of the lentivector (transfer, envelope, and packaging plasmids) is a common and flexible means of delivering genetic material in laboratory settings. However, scaling-up transient transfections for clinical applications is cumbersome and may lead to the development of replication-competent lentivirus 5,6. To overcome these hurdles, several stable packaging and producer cell lines have been developed 6-11. One of these lines, the GPR packaging line 11, has the attractive advantage of being regulated by tetracycline. In this paper, we demonstrate how to adapt this system to produce self-inactivating lentiviral vectors that are specifically targeted toward dendritic cells (DC-LVs) 12.
Dendritic cells (DCs) are the most robust antigen presenting cells of the immune system. They have been the subject of great interest in cancer vaccine development because they directly initiate, program, and regulate tumor-specific immune responses 13. Incorporating a vaccination protocol to include DCs has the potential to elicit a stronger antitumor immune response than peptide or DNA vaccines. Recently, we have developed a lentiviral vector that specifically targets dendritic cells through a modified sindbis virus glycoprotein, SVGmu 14. These vectors are unique in that they show high specificity to dendritic cells and generate stronger immune responses than nonspecific, VSVg-pseudotyped vectors.
Here, we report a method for producing large quantities of these DC-targeted lentiviral vectors. We demonstrate that these DC-LVs can infect DCs and generate strong CD8+ T cell immune responses. All procedures involving animals were performed humanely, under the approval of the USC Intitutional Animal Care and Use Committee. In order to perform in vivo and clinical experiments, it is critical to create a cell line that can produce virus at a high titer. Performing the transduction and transfection steps exactly as described will maximize the chances of generating such a clone.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Mediatech Inc.</td>
			<td>10-013-CV</td>
			<td></td>
		 </tr>
		 <tr>
			<td>FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Glutamine</td>
			<td>Mediatech Inc.</td>
			<td>25-005-Cl</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Doxycycline</td>
			<td>Clontech Laboratories, Inc.</td>
			<td>631311</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Zeocin</td>
			<td>Invitrogen</td>
			<td>R250-01</td>
			<td>Toxic</td>
		 </tr>
		 <tr>
			<td>Puromycin</td>
			<td>Sigma-Aldrich</td>
			<td>P8833</td>
			<td></td>
		 </tr>
		 <tr>
			<td>T4 DNA Ligase</td>
			<td>NEB</td>
			<td>M0202S</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DNeasy Blood &amp; Tissue Kit</td>
			<td>Qiagen</td>
			<td>69506</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

a tetracycline-regulated cell line produces high-titer lentiviral vectors that specifically target dendritic cells

Lentiviral vectors (LVs) are a powerful means of delivering genetic material to many types of cells. Because of safety concerns associated with these HIV-1 derived vectors, producing large quantities of LVs is challenging. In this paper, we report a method for producing high titers of self-inactivating LVs. We retrovirally transduce the tet-off stable producer cell line GPR to generate a cell line, GPRS, which can express all the viral components, including a dendritic cell-specific glycoprotein, SVGmu. Then, we use concatemeric DNA transfection to transfect the LV transfer plasmid encoding a reporter gene GFP in combination with a selectable marker. Several of the resulting clones can produce LV at a titer 10-fold greater than what we achieve with transient transfection. Plus, these viruses efficiently transduce dendritic cells in vitro and generate a strong T cell immune response to our reporter antigen. This method may be a good option for producing strong LV-based vaccines for clinical studies of cancer or infectious diseases.

The stable cell line described in this method can produce large quantities of lentiviral vectors that are specifically targeted to dendritic cells. As shown in Figure 1B, isolation of individual clones yielded stable cell lines of varying quality 12. Among 26 clones tested, 8 produced lentiviral particles at a titer of greater than 106 transduction units per ml (TU/ml), which is a typical benchmark for SVG-pseudotyped lentiviral vectors produced by transient transfection. At the sam...

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

Here, we use retroviral transduction and concatemeric transfection to create a cell line that can express the components of a lentiviral vector (LV) in the absence of tetracycline. This LV encodes GFP and is pseudotyped with a glycoprotein, SVGmu, which is specific for a receptor on dendritic cells.

Lentiviral  vectors (LVs) are a powerful means of delivering genetic material to many types  of cells. Because of safety concerns associated with these ...

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14.4K Views.  University of Southern California, Los Angeles. Here, we use retroviral transduction and concatemeric transfection to create a cell line that can express the components of a lentiviral vector (LV) in the absence of tetracycline. This LV encodes GFP and is pseudotyped with a glycoprotein, SVGmu, which is specific for a receptor on dendritic cells.