Name: an efficient method for adenovirus production
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This work was supported by a Project co-financed from the European Regional Development Fund through the Competitiveness Operational Program 2014-2020 (POC-A.1-A.1.1.4-E-2015, ID: P_37_668; acronym DIABETER), a grant of the Romanian Ministry of Research and Innovation PCCDI- UEFISCDI, Project number PN-III-P1-1.2-PCCDI-2017-0697 within PNCD III and by the Romanian Academy. The authors thank Kyriakos Kypreos (University of Patras, Greece) for his generous and relevant advice, Ovidiu Croitoru (University of Fine Arts, Bucharest, Romania) for filming, film editing, and graphical design, and Mihaela Bratu for technical assistance.

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The authors have nothing to disclose.

Recombinant adenoviruses are a versatile tool for gene delivery and expression12,13,14. To induce strong protein expression by adenoviral transduction, the encoding sequence of the gene of interest is inserted in the genome of the adenovirus. The AdEasy adenoviral system, developed in the laboratory of Bert Vogelstein, comprises a backbone plasmid (pAdEasy-1) containing most of the wild-type adenovirus serotype 5 genome, and a shuttle vector (pAdTrack), designed for gene cloning2,10. The deletion of the adenoviral genes E1 (responsible for the assembly of infectious virus particles) and E3 (encoding proteins involved in evading host immunity) created a space in the adenoviral genome, in which a gene of interest of 6.5-7.5 kb can be inserted2,3. This size is sufficient for many genes, especially for those with shorter introns15,16,17. There are also researchers reporting the production of adenoviruses carrying the cDNA of a transgene18,19,20. However, we obtained a lower yield of transgene expression for cDNA-carrying adenoviruses than for their counterparts carrying a gene or a mini-gene (data not shown).
Improving and adapting the previous methods2,10,14,18,21, the technology for adenoviral production requires a shorter time, lower cost, and less effort. The full-length adenoviral DNA is obtained by recombination between the shuttle vector and the pAdEasy-1 plasmid in the homologous recombination prone E. coli strain, BJ5183. The protocol implies the chemical transformation of AdEasier-1 cells (BJ5183 bacteria containing pAdEasy-1). This technique does not require an electroporator that may not be available in some laboratories, is very simple, increases the recombination yield, and reduces the time necessary to obtain competent cells and to perform the transformation. The preselection of recombinant clones performed by PCR further shortens the time and eases the whole procedure. A similar procedure was used by Zhao and co-workers22, however, in the protocol, we optimized the sequences of the primers.
For the GFP-adenovirus packaging and amplification, a HEK293 derivative cell line was used, namely AD293 cells, which are more adherent to the culture plate. Other cell lines commonly used for adenoviral production are the following: 911, 293FT, pTG6559 (A549 derivative), PER.C6 (HER derivative), GH329 (HeLa derivative), N52.E6, and HeLa-E123,24,25,26. In our hands, no improvement in the adenoviral production was obtained when 911 cells were used (data not shown). The transfection of AD293 cells with the recombinant plasmid using K2 reagent highly increased the efficiency of the viral packaging step. After adenovirus production, up to ~70% of the adenovirus is still inside the cells and is released by three freezing and thawing cycles. Increasing the number of cycles is not suitable because it destroys the adenovirus.
Throughout the routine adenoviral production process, numerous viral particles are released in the cell culture medium. Discarding this cell culture medium during the harvesting of the infected AD293 cells would result in an important viral loss. We optimized the protocol described by Schagen and co-workers to purify the adenoviral particles from the cell culture medium by precipitation with ammonium sulfate27. This method has a higher efficiency in adenovirus recovery from the cell culture medium as compared with the method using polyethylene glycol28. The precipitated adenovirus should be purified immediately by ultracentrifugation or kept in the refrigerator for a couple of days but only after dialysis, to remove the salt excess. Keeping the precipitate longer than a few hours without dialysis is harmful to the virus.
Purification of the adenoviral particles by ultracentrifugation performed in one-step reduces the manipulation of the adenoviral stock and eases the procedure as compared with the protocols using successive ultracentrifugation steps14,29. Dialysis of the purified adenovirus is necessary to remove cesium chloride that may further affect transduction. In the protocol, we used Tris buffer containing MgCl2 but not sucrose for dialysis, since it requires a huge, unjustified amount of sucrose that is needed otherwise as a preservative for freezing. Thus, we added sucrose later, directly into the adenoviral stocks prepared for freezing. To avoid frequent freezing and thawing of the purified adenovirus, it is advisable to aliquot the adenoviral stocks and to store them at -80 &#176;C. The adenoviral titer was evaluated by flow cytometry considering the GFP reporter gene and the percentage of transduced cells for a specific viral dilution. This method is faster as compared with the classical &#8220;plaque assay&#8221; and is more trustful as compared with the evaluation of the capsid proteins (by various methods such as ELISA or flow cytometry) which does not reveal the capacity of infection of the adenoviral particles. However, ELISA-based quantification, Q-PCR, or plaque assay using commercially available kits are alternative methods, especially useful for titration of adenoviruses which do not contain a fluorescent tracer.
Considering that pAdTrack adenoviruses are derived from human adenoviruses serotype 5 which is recognized by Coxsackievirus and Adenovirus Receptors (CAR), we demonstrated the capacity of the GFP-adenovirus to transduce cells of human origin (endothelial cells), but also cells of other origins: bovine (endothelial cells) and murine (mesenchymal stromal cells and hepatocytes). The data showed that the GFP-adenovirus can induce a high level of expression of a transgene.
In conclusion, we optimized this laborious technology to reduce the time, the costs, and the effort needed to obtain the adenoviral particles. The adenovirus prepared is able to infect various cell types and to induce the expression of the gene of interest. This protocol may be used in a variety of experiments since the adenoviral-mediated gene transfer represents one of the main tools for developing modern gene therapies.
ABBREVIATIONS: AdV-GFP, adenoviral particles; BAEC, bovine aortic endothelial cells; CsCl, cesium chloride; GFP, green fluorescent protein; MSC, mesenchymal stromal cells; TU, transducing units.

Adenoviruses are nonenveloped viruses containing a nucleocapsid and a double-stranded linear DNA genome1,2,3. Adenoviruses can infect a broad range of cell types and infection is not dependent on active host cell division. After infection, the adenovirus introduces its genomic DNA into the host cell nucleus, where it stays epichromosomal and is transcribed together with the genes of the host. Thus, a minimal potential risk for insertional mutagenesis or oncogenes regulation is attained4,5,6. The adenoviral genome is not replicated together with the host genome and thus, the adenoviral genes are diluted in a dividing cell population. Among the advantages of adenoviral transduction, there are: (i) high levels of transgene expression; (ii) reduced risks related to the integration of the viral DNA into the host genome, due to episomal expression; (iii) transduction of a wide variety of dividing and non-dividing cell types. Most adenoviruses used in biomedical research are non-replicative, lacking the E1 region7,8,9. For their production, a cell line supplying the E1 sequence (such as HEK293) is required. Besides, a non-essential region for the viral life cycle (E3) was deleted to allow the insertion of a transgene in the viral genome; other regions (E2 and E4) were further deleted in some adenoviruses, but in these cases, a decreased yield of adenoviral production and low expression of the transgene were reported7.
Here, we present an improved protocol for constructing, packaging, and purifying the adenoviruses using the AdEasy System. These improvements allowed the packing of the adenovirus in a faster and more economical way as compared to the original method developed by Bert Vogelstein2,10, due to the following advantages: (i) the recombination in BJ5183-containing pAdEasy-1 by chemical transformation of bacteria; (ii) the selection of the recombinant clones by PCR; (iii) the transfection of AD293 cells using the K2 transfection system for adenoviral packaging; (iv) the precipitation of adenoviral particles from culture medium after viral packaging and amplification; (v) the adenoviral purification using one-step cesium chloride (CsCl) gradient ultracentrifugation.
The protocol for adenovirus production using the AdEasy system (Figure 1) comprises the following steps:
(1) Recombination of pAdTrack-GFP with pAdEasy-1 in BJ5183 bacteria 
(2) Packaging of the adenoviral particles 
(3) Amplification of the adenovirus 
(4) Purification of the adenoviral particles from cell lysate and culture medium 
(5) Adenovirus titration.
<img alt="Plasmid recombination and amplification diagram; pAdTrack-GFP process with PCR, cell transfection." class="xfigimg" src="/files/ftp_upload/61691/61691fig01.jpg" data-altupdatedat="1747911167000" data-alt="Gene editing diagram of pAdTrack-GFP process; includes recombination, packaging, amplification steps."> 
Figure 1: The adenovirus production technology. The main steps of the adenoviral technology are: (1) The recombination of the pAdTrack-GFP with the pAdEasy-1 plasmid in BJ5183 bacteria. The selected recombined plasmids are amplified in DH5α bacteria and then purified; (2) The packaging of the adenoviral particles in AD293 cells, that are producing adeno-E1 proteins; (3) The amplification of the adenovirus in AD293 cells; (4) The purification of the adenoviral particles from the cell lysate and the culture medium by ultracentrifugation on a CsCl density gradient; (5) The titration of the adenovirus and the functional testing. <a href="https://www.jove.com/files/ftp_upload/61691/61691fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In this protocol, we exemplified the technology for the production of the adenovirus, which can induce the expression of GFP in the host cells. GFP is already inserted in the backbone of the pAdTrack-CMV shuttle vector (Addgene #16405), under a second CMV promoter and is used as a reporter gene (Figure 1). For this reason, here we designated the pAdTrack-CMV vector as pAdTrack-GFP and we assessed the expression of GFP for demonstrative purposes. Besides GFP expression, the system can be used to overexpress a gene of interest, which may be cloned in the multiple cloning sites of the pAdTrack-CMV. A gene or a minigene cloned in the pAdTrack-CMV is usually more efficient for expression induction as compared with the cDNA11. The data showed a strong GFP expression in transduced cells (such as hepatocytes, endothelial cells) from various sources (human, bovine, murine). Adenoviral-mediated gene transfer represents one of the main tools for developing modern gene therapies.

<table><tbody><tr><td>AD293 cells</td><td>Agilent Technologies</td><td>240085</td><td></td></tr><tr><td>AdEasier-1 cells</td><td>Addgene</td><td>16399</td><td></td></tr><tr><td>Agarose I (for electrophoresis)</td><td>Thermo Scientific</td><td>17850</td><td></td></tr><tr><td>Ammonium sulfate</td><td>Sigma</td><td>A4418</td><td></td></tr><tr><td>Ampicillin sodium salt</td><td>Sigma</td><td>A0166</td><td></td></tr><tr><td>BamH I</td><td>Thermo Scientific</td><td>FD0054</td><td></td></tr><tr><td>Cell culture plates 100 mm</td><td>Eppendorf</td><td>30702115</td><td></td></tr><tr><td>Cesium chloride</td><td>Sigma</td><td>L4036</td><td></td></tr><tr><td>DH5alpha bacteria</td><td>Thermo Scientific</td><td>18265017</td><td></td></tr><tr><td>DMEM (GlutaMAX, 4.5g/L D-Glucose)</td><td>Gibco</td><td>3240-027</td><td></td></tr><tr><td>EA.hy926 cells</td><td>ATCC</td><td>CRL-2922</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>E5134</td><td></td></tr><tr><td>Ethanol (99.8%)</td><td>Roth</td><td>5054.2</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma</td><td>F7524</td><td></td></tr><tr><td>Flasks T25, T75, T175</td><td>Eppendorf</td><td>30712129</td><td></td></tr><tr><td>Glucose</td><td>Sigma</td><td>G7021</td><td></td></tr><tr><td>Hepa 1-6 murine hepatocytes</td><td>ATCC</td><td>CRL-1830</td><td></td></tr><tr><td>Hind III</td><td>Thermo Scientific</td><td>FD0504</td><td></td></tr><tr><td>Kanamycin Sulfate</td><td>Thermo Scientific</td><td>15160054</td><td></td></tr><tr><td>K2 Transfection System</td><td>Biontex</td><td>T060-5.0</td><td></td></tr><tr><td>LB medium</td><td>Formedium</td><td>LBx0102</td><td></td></tr><tr><td>LB-agar</td><td>Formedium</td><td>LBx0202</td><td></td></tr><tr><td>Mix &amp;amp; Go &lt;em&gt;E. coli&lt;/em&gt; Transformation kit</td><td>Zymo Research</td><td>T3001</td><td></td></tr><tr><td>Midori Green Advanced DNA stain</td><td>Nippon Genetics Europe</td><td>MG-04</td><td></td></tr><tr><td>NaOH</td><td>Sigma</td><td>S8045</td><td></td></tr><tr><td>Opti-MEM</td><td>Thermo Scientific</td><td>31985070</td><td></td></tr><tr><td>Pac I</td><td>Thermo Scientific</td><td>FD2204</td><td></td></tr><tr><td>pAdEasy-1</td><td>Addgene</td><td>16400</td><td></td></tr><tr><td>pAdTrack-CMV</td><td>Addgene</td><td>16405</td><td></td></tr><tr><td>Phenol:chloroform:isoamyl alcohol (24:24:1)</td><td>Invitrogen</td><td>15593-031</td><td></td></tr><tr><td>Polymerase GoTaq</td><td>Promega</td><td>M3005</td><td></td></tr><tr><td>Pme I (Mss I)</td><td>Thermo Scientific</td><td>FD1344</td><td></td></tr><tr><td>Potassium acetate</td><td>VWR Chemicals</td><td>43065P</td><td></td></tr><tr><td>Pst I</td><td>Thermo Scientific</td><td>FD0614</td><td></td></tr><tr><td>Qiagen Midi Prep kit</td><td>Qiagen</td><td>12125</td><td></td></tr><tr><td>Cell Scraper</td><td>TPP</td><td>99003</td><td></td></tr><tr><td>SDS</td><td>Thermo Scientific</td><td>28365</td><td></td></tr><tr><td>Slide-A-Lyzer dialysis cassettes</td><td>Thermo Scientific</td><td>66330</td><td></td></tr><tr><td>Sodium pyruvate</td><td>SIGMA</td><td>P5280-100G</td><td></td></tr><tr><td>Syringe with 23G neeedle</td><td>B Braun</td><td>464BR</td><td></td></tr><tr><td>Tris HCl</td><td>Sigma</td><td>1185-53-1</td><td></td></tr><tr><td>Trypan blue</td><td>Roth</td><td>CN76.1</td><td></td></tr><tr><td>Tubes 50ml</td><td>TPP</td><td>91050</td><td></td></tr><tr><td>Ultra-Clear Tubes (14x89 mm)</td><td>Beckman Coulter</td><td>344059</td><td></td></tr><tr><td>Centrifuge (refrigerated)</td><td>Sigma Sartorius</td><td>3-19KS</td><td></td></tr><tr><td>HeraeusFresco 17 Microcentrifuge</td><td>Thermo Scientific</td><td>75002420</td><td></td></tr><tr><td>Ultracentrifuge with SW41Ti rotor</td><td>Beckman Coulter</td><td>Optima L-80 XP</td><td></td></tr><tr><td>Culture Hood</td><td>Thermo Scientific</td><td>Class II</td><td></td></tr><tr><td>Pipettes (0-2&amp;micro;l, 1-10&amp;micro;l, 2-20&amp;micro;l, 10-100&amp;micro;l, 20-200&amp;micro;l, 100-1000&amp;micro;l)</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Dry Block Heating Thermostat</td><td>Biosan</td><td>TDB-120</td><td></td></tr><tr><td>Thermocycle</td><td>SensoQuest</td><td>012-103</td><td></td></tr><tr><td>Water Bath</td><td>Memmert</td><td>WNB 14</td><td></td></tr></tbody></table>

an efficient method for adenovirus production

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types and organs. However, recombinant adenoviral technology is laborious, time-consuming, and expensive. Here, we present an improved protocol using the pAdEasy system to obtain purified adenoviral particles that can induce a strong green fluorescent protein (GFP) expression in transduced cells. The advantages of this improved method are faster preparation and decreased production cost compared to the original method developed by Bert Vogelstein. The main steps of the adenoviral technology are: (1) the recombination of pAdTrack-GFP with the pAdEasy-1 plasmid in BJ5183 bacteria; (2) the packaging of the adenoviral particles; (3) the amplification of the adenovirus in AD293 cells; (4) the purification of the adenoviral particles from cell lysate and culture medium; and (5) the viral titration and functional testing of the adenovirus. The improvements to the original method consist of (i) the recombination in BJ5183-containing pAdEasy-1 by chemical transformation of bacteria; (ii) the selection of recombinant clones by &#8220;negative&#8221; and &#8220;positive&#8221; PCR; (iii) the transfection of AD293 cells using the K2 transfection system for adenoviral packaging; (iv) the precipitation with ammonium sulfate of the viral particles released by AD293 cells in cell culture medium; and (v) the purification of the virus by one-step cesium chloride discontinuous gradient ultracentrifugation. A strong expression of the gene of interest (in this case, GFP) was obtained in different types of transduced cells (such as hepatocytes, endothelial cells) from various sources (human, bovine, murine). Adenoviral-mediated gene transfer represents one of the main tools for developing modern gene therapies.

We modified and improved the original Vogelstein’s protocol in order to attain faster and more efficient adenovirus production. First, we revised the methodology to achieve an easier selection of recombinants. After recombination, the BJ5183 bacterial clones were tested by “negative PCR” to assess the integrity of pAdTrack-GFP as an indicator of the lack of recombination (Figure 3A), or by “positive PCR” to identify the gene of interest, assimilated in our case to GFP (Figure 3B). In both “negative” and “positive” PCRs, we used pAdTrack-GFP as a control template, which gave a band of 986 bp for pAdTrack integrity (Figure 3A, lane 1), and a band of 264 bp for GFP (Figure 3B, lane 3). The primers used for the “negative PCR” were designed to amplify a fragment of 986 bp containing the PmeI site in pAdTrack-GFP. This DNA fragment is drastically enlarged after recombination and is not amplified in the positive recombinant clones. Negative clones for recombination, in which pAdTrack-GFP remained intact, are represented in Figure 3A, lanes 3, 4, and 6. The primers anneal on the DNA sequences adjacent to the recombination site. Potential positive recombinant clones (Figure 3A, lanes 2 and 5) expressed GFP as shown in Figure 3B, lane 1, and 2. Plasmid DNA from these clones was isolated and used for DH5α transformation to obtain a higher amount of DNA. These preselected recombinant plasmids amplified in DH5α were then tested by enzymatic digestion. In Figure 3C-E are illustrated the results of the enzymatic digestion of one recombinant-positive clone digested with Hind III, PstI, BamHI restriction enzymes (Figure 3C, D, E lane 2). The HindIII and PstI digestion patterns of the recombinant clone were similar to those obtained for pAdEasy-1 since HindIII and PstI cut the pAdEasy-1 plasmid 24 and 25 times, respectively, (Figure 3C and D, lane 3); HindIII cut once and PstI cut four times the pAdTrack-GFP vector (Figure 3C and D, lane 1). BamHI cut twice pAdEasy-1 vector (Figure 3C, lane 3), and once pAdTrack-GFP (Figure 3C, lane 1).
PacI cut out a fragment of 4.5 kb from the recombinant plasmid (Figure 3F, lane 2), a fragment of 2863 bp from pAdTrack-GFP (Figure 3F, lane 1), and linearized the pAdEasy-1 vector (Figure 3F, lane 3). The DNA ladder is represented in Figure 3C-F, in lanes 4. The recombinant plasmid was digested with Pac I for further use for AD293 transfection.
<img alt="Recombinant plasmid selection by PCR, gel electrophoresis of enzymatic digestion, DNA fragment sizes." class="xfigimg" src="/files/ftp_upload/61691/61691fig03v2.jpg" data-altupdatedat="1747911167000" data-alt="PCR selection and enzymatic digestion gel electrophoresis of recombinant plasmids using HindIII, PstI."> 
Figure 3: The recombination of pAdTrack-GFP with the pAdEasy-1 plasmid. The plasmids obtained after the recombination of pAdTrack-GFP and pAdEasy-1 were tested by “negative” PCR for the pAdTrack-GFP integrity (A). The non-recombinant clones were evidenced by the presence of a 986 bp band corresponding to the sequence amplified from the pAdTrack-GFP plasmid (A, lanes 3, 4, and 6). The clones potentially positive for recombination (A, lanes 2 and 5) were also obtained. When the pAdTrack-GFP vector was used as a template, a band of 986 bp for pAdTrack-GFP (A, lane 1) was obtained. The potentially positive recombinant clones were tested for GFP expression by “positive” PCR (B); a band of 264 bp appears for both potentially recombined clones (B, lane 1 and 2), as well as for the pAdTrack-GFP plasmid. The DNA from one potential recombinant clone was tested with HindIII, PstI, BamHI, and PacI restriction enzyme (C-F, lanes 2). In the controls, the pAdEasy-1 vector (C-F, lanes 3) and the pAdTrack-GFP plasmid (C-F, lanes 1) were digested with the same enzymes. The DNA ladder is represented in C-F lane 4. <a href="https://www.jove.com/files/ftp_upload/61691/61691fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The adenoviral packaging and amplification were performed in AD293 cells. The adenoviral particles (AdV-GFP) were purified from the AD293 cell lysate as well as from the cell culture medium, where they had been released by the infected cells. To concentrate the adenovirus found in the cell culture medium, the particles were precipitated with ammonium sulfate and then resuspended in 10 mM Tris HCl pH 8 with 2 mM MgCl2, the same buffer as that used for cell lysis. Subsequently, the adenoviral particles from the cell lysate and from the culture medium were purified by CsCl discontinuous gradient ultracentrifugation. After ultracentrifugation, a strong band of purified AdV-GFP was obtained, as shown in Figure 4.
<img alt="Cell lysate and culture medium density gradient centrifugation tubes; adenoviral band separation." class="xfigimg" src="/files/ftp_upload/61691/61691fig04.jpg" data-altupdatedat="1747911167000" data-alt="Cell lysate and culture medium separation in CsCl density gradient centrifugation, adenoviral band visible."> 
Figure 4: The adenoviral purification by ultracentrifugation on a discontinuous CsCl gradient. The cell homogenate and the adenovirus precipitated from the medium were subjected to ultracentrifugation on a discontinuous gradient formed by low and high-density CsCl solutions. Strong bands of GFP- adenovirus were evidenced in both cases. <a href="https://www.jove.com/files/ftp_upload/61691/61691fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To determine the viral titer expressed in transducing units per one mL (TU/mL), the AD293 cells were infected with serial dilutions of the AdV-GFP. After 48 hours, the infected cells expressed GFP, in an inverse correlation with the dilution factor of the viral suspension. This was observed by fluorescence microscopy and the percentage of GFP-positive cells was determined by flow cytometry (Figure 5). To calculate the titer, the viral dilution that induced 5 - 20% of GFP-positive cells was considered (Figure 5C). Usually, we obtain a viral titer of ~1010 (TU/mL) for GFP-adenovirus.
Below, we provide an example of an adenoviral titer calculation for a specific adenoviral batch in which 300000 cells (C) were transduced with 1 mL adenoviral solution (V), at a dilution factor of 106 (D), for which 6% GFP-positive cells (F) were obtained:
Titer (TU/mL) = D x F/100 x C/V = 106 x 6/100 x 300000/1 = 1.8 x 1010 TU/mL
<img alt="Dilution series fluorescence microscopy and flow cytometry analysis for GFP expression." class="xfigimg" src="/files/ftp_upload/61691/61691fig05.jpg" data-altupdatedat="1747911167000" data-alt="Fluorescence microscopy of GFP dilution; flow cytometry graphs; transduction efficiency analysis."> 
Figure 5: The assessment of the adenoviral titer. AD293 cells were infected with various adenoviral dilutions. Forty-eight hours later, the cells were observed by fluorescence microscopy and analyzed by flow cytometry to determine the percentage of GFP positive cells induced by different adenoviral dilutions (A-D). To establish the gate for flow cytometry, non-transduced cells were also analyzed (E). The titer calculated for the dilution factor 106, when 6% of the cells were GFP positive was 1.8 x 1010 TU/mL. For panels A-E, bars: 100µm. <a href="https://www.jove.com/files/ftp_upload/61691/61691fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To test the transduction potential of the prepared adenovirus, four cell lines were used: human endothelial cells (EA.hy926), bovine aortic endothelial cells (BAEC), murine hepatocytes (Hepa 1-6), and murine mesenchymal stromal cells (MSC). Endothelial cells (EA.hy926 and BAEC) were transduced with 25 TU/cell, the hepatocytes were transduced with 5 TU/cell and MSC were transduced with 250 TU/cell.
Here is an example of how the volume of adenoviral suspension needed to infect 3 x 106 cells with 25 TU/cell, using the adenoviral suspension with 1.8 x 1010 TU/mL, was calculated.
For 1 cell .............. 25 TU 
3 x 106 cells .............. x TU <img alt="DNA transcription process; RNA polymerase action; biochemical pathway diagram; gene expression model." src="/files/ftp_upload/61691/61691equ01.jpg" data-altupdatedat="1747911167000" data-alt="Static equilibrium ΣFx=0 diagram; friction, tension concepts."> x=75 x 106 TU 
If the viral stock contains 
1.8 x 1010 TU .............. 1 mL 
75 x 106 TU .............. y mL <img alt="DNA transcription process; RNA polymerase action; biochemical pathway diagram; gene expression model." src="/files/ftp_upload/61691/61691equ01.jpg" data-altupdatedat="1747911167000" data-alt="Static equilibrium ΣFx=0 diagram; friction, tension concepts."> y= 4.2 x 10-3 mL = 4.2µL of viral stock
Forty-eight hours after transduction, the cells were analyzed by fluorescence microscopy. As shown in Figure 6, human or bovine endothelial cells were transduced with good efficiency (~50%) for 25 TU/cell (Figure 6 EA.hy926 and BAEC). Murine hepatocytes (Hepa 1-6) were efficiently transduced by the adenovirus at a low amount of adenovirus particles (5 TU/cell), but they are also sensitive to the adenovirus since a higher percentage of dead cells (PI-positive cells) was recorded (~16%) as compared to the other cell types. Mesenchymal stromal cells were the most difficult to transduce (Figure 6), due to the lack of specific adenoviral receptors (unpublished data).
<img alt="Flow cytometry and fluorescence microscopy for GFP expression analysis in various cell lines." class="xfigimg" src="/files/ftp_upload/61691/61691fig06v2.jpg" data-altupdatedat="1747911167000" data-alt="Transfection efficiency analysis via flow cytometry and fluorescence microscopy for GFP expression."> 
Figure 6: The infectivity of the adenovirus and the induction of GFP expression in transduced cells. Human endothelial cells (EA.hy926), bovine aortic endothelial cells (BAEC), murine hepatocytes (Hepa 1-6), and murine mesenchymal stromal cells (MSC) were transduced with the indicated amount of adenovirus. GFP was detected by fluorescence microscopy and the percentage of the GFP positive cells was analyzed by flow cytometry. PI-positive cells determined by flow cytometry show the cell mortality determined by viral transduction. EA.hy926 cells, bovine aortic endothelial cells, and Hepa 1-6 cells were highly transduced by the adenovirus, the yield of transduction ranging from 41 - 52%. For MSC, a higher amount of virus (250 TU/cells) induced only 27% GFP positive of the transduced cells. Bars: 100µm. <a href="https://www.jove.com/files/ftp_upload/61691/61691fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about An Efficient Method for Adenovirus Production at JoVE.com

An Efficient Method for Adenovirus Production

Here, we present a protocol for adenovirus production using the pAdEasy system. The technology includes the recombination of the pAdTrack and pAdEasy-1 plasmids, the adenovirus packaging and amplification, the purification of the adenoviral particles from cell lysate and culture medium, the viral titration, and the functional testing of the adenovirus.

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types ...

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Bioengineering

13.0K Views.  Institute of Cellular Biology and Pathology "Nicolae Simionescu". Here, we present a protocol for adenovirus production using the pAdEasy system. The technology includes the recombination of the pAdTrack and pAdEasy-1 plasmids, the adenovirus packaging and amplification, the purification of the adenoviral particles from cell lysate and culture medium, the viral titration, and the functional testing of the adenovirus.

Video: An Efficient Method for Adenovirus Production

Production and Titering of Recombinant Adeno-associated Viral Vectors

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced.

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

Immunology and Infection

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 which regulate the expression of non-structural (NS1 and NS2) and capsid proteins (VP1 and VP2) respectively1. They attract high interest as anticancer agents for their oncolytic and oncosuppressive abilities while being non-pathogenic for humans2. NS1 is the major effector of viral cytotoxicity3. In order to further enhance their natural antineoplastic activities, derivatives from these vectors have been generated by replacing the gene encoding for the capsid proteins with a therapeutic transgene (e.g. a cytotoxic polypeptide, cytokine, chemokine, tumour suppressor gene etc.)4. The recombinant parvoviruses (recPVs) vector retains the NS1/2 coding sequences and the PV genome telomeres which are necessary for viral DNA amplification and packaging. Production of recPVs occurs only in the producer cells (generally HEK293T), by co-transfecting the cells with a second vector (pCMV-VP) expressing the gene encoding for the VP proteins (Fig. 1)4. The recPV vectors generated in this way are replication defective. Although recPVs proved to possess enhanced oncotoxic activities with respect to the parental viruses from which they have been generated, their production remains a major challenge and strongly hampers the use of these agents in anti-cancer clinical applications.
We found that introduction of an Ad-5 derived vector containing the E2a, E4(orf6) and the VA RNA genes (e.g. pXX6 plasmid) into HEK293T improved the production of recPVs by more than 10 fold in comparison to other protocols in use. Based on this finding, we have constructed a novel Ad-VP-helper that contains the genomic adenoviral elements necessary to enhance recPVs production as well as the parvovirus VP gene unit5. The use of Ad-VP-helper, allows production of rec-PVs using a protocol that relies entirely on viral infection steps (as opposed to plasmid transfection), making possible the use of cell lines that are difficult to transfect (e.g. NB324K) (Fig. 2). We present a method that greatly improves the amount of recombinant virus produced, reducing both the production time and costs, without affecting the quality of the final product5. In addition, large scale production of recPV (in suspension cells and bioreactors) is now conceivable.

Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Production and Purification of Non Replicative Canine Adenovirus Type 2 Derived Vectors

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the frequent pre-existing immunity against the classically used human adenovirus type 5, canine adenovirus type 2 (CAV2) has been proposed as an alternative vector for human gene transfer. The well-characterized biology of CAV2, together with its ease of genetic manipulation, offer major advantages, notably for gene transfer into the central nervous system, or for inducing a wide range of protective immune responses, from humoral to cellular immunity. Nowadays, CAV2 represents one of the most appealing nonhuman adenovirus for use as a vaccine vector. This protocol describes a simple method to construct, produce and titer recombinant CAV2 vectors. After cloning the expression cassette of the gene of interest into a shuttle plasmid, the recombinant genomic plasmid is obtained by homologous recombination in the E. coli BJ5183 bacterial strain. The resulting genomic plasmid is then transfected into canine kidney cells expressing the complementing CAV2-E1 genes (DK-E1). A viral amplification enables the production of a large viral stock, which is purified by ultracentrifugation through cesium chloride gradients and desalted by dialysis. The resulting viral suspension routinely has a titer of over 1010 infectious particles per ml and can be directly administrated in vivo.

These past 15 years, canine adenovirus type 2 (CAV2)-derived vectors have proven their efficiency to transduce cells in vitro&#160;and in vivo and are widely used for vaccination and gene therapy. Here, we describe a procedure to construct, produce and purify CAV2 vectors, giving rise to high-titer viral suspensions.

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the ...

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity, and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks, and shortcuts developed over the past years working with this HCAdV vector system are presented.

A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans.

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high ...

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

Biology

Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these traditionally modified Ad5 vectors in clinical trials could be primarily correlated with Ad5 pre-existing immunity (PEI) among the majority of the population. To promote Ad5-vectored vaccine development by solving the concern of Ad5 PEI, the innovative Antigen Capsid-Incorporation strategy has been employed. By merit of this strategy, Ad5-vectored we first constructed the hexon shuttle plasmid HVR1-KWAS-HVR5-His6/pH5S by subcloning the hypervariable region (HVR) 1 of hexon into a previously constructed shuttle plasmid HVR5-His6/pH5S, which had His6 tag incorporated into the HVR5. This HVR1 DNA fragment containing a HIV epitope ELDKWAS was synthesized. HVR1-KWAS-HVR5-His6/pH5S was then linearized and co-transformed with linearized backbone plasmid pAd5/&#8710;H5 (GL) , for homologous recombination. This recombined plasmid pAd5/H5-HVR1-KWAS-HVR5-His6 was transfected into cells to generate the viral vector Ad5/H5-HVR1-KWAS-HVR5-His6. This vector was validated to have qualitative fitness indicated by viral physical titer (VP/ml), infectious titer (IP/ml) and corresponding VP/IP ratio. Both the HIV epitope and His6 tag were surface-exposed on the Ad5 capsid, and retained epitope-specific antigenicity of their own. A neutralization assay indicated the ability of this divalent vector to circumvent neutralization by Ad5-positive sera in vitro. Mice immunization demonstrated the generation of robust humoral immunity specific to the HIV epitope and His6. This proof-of-principle study suggested that the protocol associated with the Antigen Capsid-Incorporation strategy could be feasibly utilized for the generation of Ad5-vectored vaccines by modifying different capsid proteins. This protocol could even be further modified for the generation of rare-serotype adenovirus-vectored vaccines.

Here, we present a protocol to generate a proof-of-principle divalent adenovirus type 5 (Ad5) vector Ad5/H5-HVR1-KWAS-HVR5-His6 by utilizing the Antigen Capsid-Incorporation strategy. This vector was demonstrated to exhibit qualitative fitness, the capability to escape Ad5-positive sera in vitro, and the antigenicity as well as immunogenicity to the incorporated antigens.

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these ...

Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically narrowed segments of coronary and peripheral arteries. Endovascular stents capable of tunable release of genes with anti-restenotic activity may present an alternative strategy to presently used drug-eluting stents. In order to attain clinical translation, gene-eluting stents must exhibit predictable kinetics of stent-immobilized gene vector release and site-specific transduction of vasculature, while avoiding an excessive inflammatory response typically associated with the polymer coatings used for physical entrapment of the vector. This paper describes a detailed methodology for coatless tethering of adenoviral gene vectors to stents based on a reversible binding of the adenoviral particles to polyallylamine bisphosphonate (PABT)-modified stainless steel surface via hydrolysable cross-linkers (HC). A family of bifunctional (amine- and thiol-reactive) HC with an average t1/2 of the in-chain ester hydrolysis ranging between 5 and 50 days were used to link the vector with the stent. The vector immobilization procedure is typically carried out within 9 hr&#160;and consists of several steps: 1) incubation of the metal samples in an aqueous solution of PABT (4 hr); 2) deprotection of thiol groups installed in PABT with tris(2-carboxyethyl) phosphine (20 min); 3) expansion of thiol reactive capacity of the metal surface by reacting the samples with polyethyleneimine derivatized with pyridyldithio (PDT) groups (2 hr); 4) conversion of PDT groups to thiols with dithiothreitol (10 min); 5) modification of adenoviruses with HC (1 hr); 6) purification of modified adenoviral particles by size-exclusion column chromatography (15 min) and 7) immobilization of thiol-reactive adenoviral particles on the thiolated steel surface (1 hr). This technique has wide potential applicability beyond stents, by facilitating surface engineering of bioprosthetic devices to enhance their biocompatibility through the substrate-mediated gene delivery to the cells interfacing the implanted foreign material.

These studies report on reversible attachment of adenoviral gene vectors to coatless metal surfaces of stents and model mesh disks. Sustained release of transduction-competent viral particles contingent upon hydrolysis of cross-linkers used for vector immobilization results in a durable site-specific transgene expression in vascular cells and in stented arteries.

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically ...

Medicine

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments through the recruitment of viral and cellular proteins to sites occupied by the viral genome. These sites, called replication compartments (RC), can be considered viral-induced nuclear domains where the viral genome is localized and viral and cellular proteins that participate in replication, transcription and post-transcriptional processing are recruited. Moreover, cellular proteins involved in the antiviral response, such as tumor suppressor proteins, DNA damage response (DDR) components and innate immune response factors are also co-opted to RC. Although RC seem to play a crucial role to promote an efficient and productive replication cycle, a detailed analysis of their composition and associated activities has not been made. To facilitate the study of adenoviral RC and potentially those from other DNA viruses that replicate in the cell nucleus, we adapted a simple procedure based on velocity gradients to isolate Ad RC and established a cell-free system amenable to conduct morphological, functional and compositional studies of these virus-induced subnuclear structures, as well as to study their impact on host-cell interactions.

We provide a novel strategy to isolate viral replication compartments (RC) from adenovirus (Ad)-infected human cells. This approach represents a cell-free system that can help to elucidate the molecular mechanisms regulating viral genome replication and expression as well as regulation of viral-host interactions established at the RC.

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments ...

Construction of Adenoviral Vectors using DNA Assembly Technology

Adenoviral vectors have been used as a gene transfer tool in gene therapy for more than three decades. Here, we introduce a protocol to construct an adenoviral vector by manipulating the genomic DNA of wild-type HAdV-7 by using a DNA assembly method. First, an infectious clone of HAdV-7, pKan-Ad7, was generated by fusing the viral genomic DNA with a PCR product from plasmid backbone, comprising of the kanamycin-resistant gene and the origin of replication (Kan-Ori), through DNA assembly. This was done by designing a pair of PCR primers, that contained ~25 nucleotides of the terminal sequence of HAdV-7 inverted terminal repeat (ITR) at the 5' end, a non-cutter restriction enzyme site for HAdV-7 genome in the middle, and a template-specific sequence for PCR priming at the 3' end. Second, an intermediate plasmid-based strategy was employed to replace the E3 region with transgene-expressing elements in the infectious clone to generate an adenoviral vector. Briefly, pKan-Ad7 was digested with dual-cutter restriction enzyme Hpa I, and the fragment containing the E3 region was ligated to another PCR product of plasmid backbone by Gibson assembly to construct an intermediate plasmid pKan-Ad7HpaI. For convenience, restriction-assembly was used to designate the plasmid cloning method of combined restriction digestion and assembly. Using restriction-assembly, the E3 genes in pKan-Ad7HpaI was replaced with a green fluorescent protein (GFP) expression cassette, and the modified E3 region was released from the intermediate plasmid and restored to the infectious clone to generate an adenoviral plasmid pKAd7-E3GFP. Finally, pKAd7-E3GFP was linearized by Pme I digestion and used to transfect HEK293 packaging cells to rescue recombinant HAdV-7 virus. To conclude, a DNA assembly-based strategy was introduced here for constructing adenoviral vectors in general laboratories of molecular biology without the need of specialized materials and instruments.

In this study, an infectious clone of human adenovirus type 7 (HAdV-7) was constructed, and an E3-deleted HAdV-7 vector system was established by modifying the infectious clone. This strategy used here can be generalized to make gene transfer vectors from other wild-type adenoviruses.

Adenoviral vectors have been used as a gene transfer tool in gene therapy for more than three decades. Here, we introduce a protocol to construct an ...