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.

<ol>
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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+experimental+glioma+by+administration+of+adenoviral+vectors+expressing+Fas+ligand.">Treatment of experimental glioma by administration of adenoviral vectors expressing Fas ligand.</a> Human Gene Therapy. 10 (10), 1641-1648 (1999).</li><li>Okuyama, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+Fas-ligand+gene+expression+in+rodent+liver+after+intravenous+injection+of+a+recombinant+adenovirus+by+the+use+of+a+Cre-mediated+switching+system.">Efficient Fas-ligand gene expression in rodent liver after intravenous injection of a recombinant adenovirus by the use of a Cre-mediated switching system.</a> Gene Therapy. 5 (8), 1047-1053 (1998).</li><li>van Dijk, K. W., Kypreos, K. E., Fallaux, F. 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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="Figure 1" class="xfigimg" src="/files/ftp_upload/61691/61691fig01.jpg" /> 
Figure 1: The adenovirus production technology.&#160;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 DH5&#945; 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.remotexs.ntu.edu.sg/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 &#38; Go E. coli 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&#181;l, 1-10&#181;l, 2-20&#181;l, 10-100&#181;l, 20-200&#181;l, 100-1000&#181;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&#8217;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 &#8220;negative PCR&#8221; to assess the integrity of pAdTrack-GFP as an indicator of the lack of recombination (Figure 3A), or by &#8220;positive PCR&#8221; to identify the gene of interest, assimilated in our case to GFP (Figure 3B). In both &#8220;negative&#8221; and &#8220;positive&#8221; 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 &#8220;negative PCR&#8221; 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 DH5&#945; transformation to obtain a higher amount of DNA. These preselected recombinant plasmids amplified in DH5&#945; 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="Figure 3" class="xfigimg" src="/files/ftp_upload/61691/61691fig03v2.jpg" /> 
Figure 3: The recombination of pAdTrack-GFP with the pAdEasy-1 plasmid.&#160;The plasmids obtained after the recombination of pAdTrack-GFP and pAdEasy-1 were tested by &#8220;negative&#8221; 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 &#8220;positive&#8221; 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.remotexs.ntu.edu.sg/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="Figure 4" class="xfigimg" src="/files/ftp_upload/61691/61691fig04.jpg" /> 
Figure 4: The adenoviral purification by ultracentrifugation on a discontinuous CsCl gradient.&#160;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.remotexs.ntu.edu.sg/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="Figure 5" class="xfigimg" src="/files/ftp_upload/61691/61691fig05.jpg" /> 
Figure 5: The assessment of the adenoviral titer.&#160;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&#160;TU/mL. For panels A-E, bars: 100&#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/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="Equation 1" src="/files/ftp_upload/61691/61691equ01.jpg" /> 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="Equation 1" src="/files/ftp_upload/61691/61691equ01.jpg" /> y= 4.2 x 10-3 mL = 4.2&#181;L 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="Figure 6" class="xfigimg" src="/files/ftp_upload/61691/61691fig06v2.jpg" /> 
Figure 6: The infectivity of the adenovirus and the induction of GFP expression in transduced cells.&#160;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: 100&#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/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 ...

an-efficient-method-for-adenovirus-production

Nanyang Technological University

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JoVE Journal

Bioengineering

12.8K 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

A Decellularization Methodology for the Production of a Natural Acellular Intestinal Matrix

Successful tissue engineering involves the combination of scaffolds with appropriate cells in vitro or in vivo. Scaffolds may be synthetic, naturally-derived or derived from tissues/organs. The latter are obtained using a technique called decellularization. Decellularization may involve a combination of physical, chemical, and enzymatic methods. The goal of this technique is to remove all cellular traces whilst maintaining the macro- and micro-architecture of the original tissue.
Intestinal tissue engineering has thus far used relatively simple scaffolds that do not replicate the complex architecture of the native organ. The focus of this paper is to describe an efficient decellularization technique for rat small intestine. The isolation of the small intestine so as to ensure the maintenance of a vascular connection is described. The combination of chemical and enzymatic solutions to remove the cells whilst preserving the villus-crypt axis in the luminal aspect of the scaffold is also set out. Finally, assessment of produced scaffolds for appropriate characteristics is discussed.

The article describes a methodology for the production of an acellular matrix from rat intestine. The derivation of intestinal scaffolds is important for future applications in tissue engineering, stem cell biology and drug testing.

Successful tissue engineering involves the  combination of scaffolds with appropriate cells in vitro or in vivo.  Scaffolds may be synthetic, ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

An Improved Mechanical Testing Method to Assess Bone-implant Anchorage

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a nano-scale. As such, traditional methods of describing implant surfaces -&#160;namely numerical determinants of surface roughness -&#160;are inadequate for predicting in vivo performance. Biomechanical testing provides an accurate and comparative platform to analyze the performance of biomaterial surfaces. An improved mechanical testing method to test the anchorage of bone to candidate implant surfaces is presented. The method is applicable to both early and later stages of healing and can be employed for any range of chemically or mechanically modified surfaces -&#160;but not smooth surfaces. Custom rectangular implants are placed bilaterally in the distal femora of male Wistar rats and collected with the surrounding bone. Test specimens are prepared and potted using a novel breakaway mold and the disruption test is conducted using a mechanical testing machine. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate and reproducible means for isolating an exact peri-implant region for testing.

An improved method to mechanically test bone anchorage to candidate implant surfaces is presented. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate means to direct the disruption forces to an exact peri-implant region.

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a ...

Production and Targeting of Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing both mono-culture and co-culture spheroids in a high throughput 96-well plate format is described in this work. This approach utilizes an aqueous two-phase system to confine cells into a drop of the denser aqueous phase immersed within the second aqueous phase. The drop rests on the well surface and keeps cells in close proximity to form a single spheroid. This technology has been adapted to a robotic liquid handler to produce size-controlled spheroids and expedite the process of spheroid production for compound screening applications. Spheroids treated with a clinically-used drug show reduced cell viability with increase in the drug dose. The use of a standard micro-well plate for spheroid generation makes it straightforward to analyze viability of cancer cells of drug-treated spheroids with a micro-plate reader. This technology is straightforward to implement both robotically and with other liquid handling tools such as manual pipettes.

A protocol for robotic printing of cancer cell spheroids in a high throughput 96-well plate format using an aqueous two-phase system is presented.

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing ...

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 ...

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

An Efficient and Reproducible Protocol for Distraction Osteogenesis in a Rat Model Leading to a Functional Regenerated Femur

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) is a surgical technique leading to bone regeneration after an osteotomy. The osteotomized extremities are moved away from each other by gradual distraction to reach the desired elongation. This procedure is widely used in humans for lower and upper limb lengthening, treatment after a bone nonunion, or the regeneration of a bone defect following surgery for bone tumor excision, as well as in maxillofacial reconstruction. Only a few studies clearly demonstrate the efficiency of their protocol in obtaining a functional regenerated bone, i.e., bone that will support physiological weight-bearing without fracture after removal of the external fixator. Moreover, protocols for DO vary and reproducibility is limited by lack of information, making comparison between studies difficult. The aim of this study was to develop a reproducible protocol comprising an appropriate external fixator design for rat limb lengthening, with a detailed surgical technique that permits physiological weight-bearing by the animal after removal of the external fixator.

This study describes a reproducible and detailed protocol using a newly developed external fixator for distraction osteogenesis (DO) in a femoral rat model which permits physiological weight-bearing by the animal after removal of the external fixator.

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive properties of in vitro transcribed (IVT) mRNA. With the help of IVT mRNA, the de novo synthesis of a desired protein can be induced without changing the physiological state of the target cell. Moreover, protein biosynthesis can be precisely controlled due to the transient effect of IVT mRNA.
For the efficient transfection of cells, nanoliposomes (NLps) may represent a safe and efficient delivery vehicle for therapeutic mRNA. This study describes a protocol to generate safe and efficient cationic NLps consisting of DC-cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as a delivery vector for IVT mRNA. NLps having a defined size, a homogeneous distribution, and a high complexation capacity, and can be produced using the dry-film method. Moreover, we present different test systems to analyze their complexation and transfection efficacies using synthetic enhanced green fluorescent protein (eGFP) mRNA, as well as their effect on cell viability. Overall, the presented protocol provides an effective and safe approach for mRNA complexation, which may advance and improve the administration of therapeutic mRNA.

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

Here we describe a protocol for the generation of cationic nanoliposomes, which is based on the dry-film method and can be used for the safe and efficient delivery of in vitro transcribed messenger RNA.

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive ...