Name: cloning and large-scale production of high-capacity adenoviral vectors based on the human adenovirus type 5
Uploaded: 2016-01-28T13:00:00
Description: Watch this Scientific Journal Video about Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5 at JoVE.com

This work was supported by DFG grant EH 192/5-1 (A.E.), the EU (E-rare-2) project Transposmart (A.E.), the UWH Forschungsf&#246;rderung (E.S. and W.Z), and the PhD programme of the University Witten/Herdecke (P.B.). J.L. was supported by a stipend of the Chinese Scholarship council and T.B. and M.G by the Else Kr&#246;ner-Fresenius foundation (EKFS).

1. Construction of Recombinant HCAdV Genomes based on the Plasmid pAdFTC
Note: All plasmids have been described previously 11,12 and are available upon request. The cloning procedure is schematically shown in Figure 1.
<ol>
	<li>Clone a gene of interest (GOI) including promoter and polyadenylation signal (pA) into the shuttle plasmid pHM5, using a cloning strategy of choice, to generate pHM5-GOI. 
	NOTE: Since pAdFTC is a relatively large plasmid, classical p...

<ol>
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J., 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=A+helper-dependent+adenovirus+vector+system:+removal+of+helper+virus+by+Cre-mediated+excision+of+the+viral+packaging+signal.">A helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal.</a> Proc Natl Acad Sci U S A. 93 (24), 13565-13570 (1996).</li><li>Palmer, D., Ng, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+system+for+helper-dependent+adenoviral+vector+production.">Improved system for helper-dependent adenoviral vector production.</a> Mol Ther. 8 (5), 846-852 (2003).</li><li>Morral, N., 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=High+doses+of+a+helper-dependent+adenoviral+vector+yield+supraphysiological+levels+of+alpha1-antitrypsin+with+negligible+toxicity.">High doses of a helper-dependent adenoviral vector yield supraphysiological levels of alpha1-antitrypsin with negligible toxicity.</a> Hum Gene Ther. 9 (18), 2709-2716 (1998).</li><li>Muruve, D. A., 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=Helper-dependent+adenovirus+vectors+elicit+intact+innate+but+attenuated+adaptive+host+immune+responses+in+vivo.">Helper-dependent adenovirus vectors elicit intact innate but attenuated adaptive host immune responses in vivo.</a> J Virol. 78 (11), 5966-5972 (2004).</li><li>Ehrhardt, A., 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=A+gene-deleted+adenoviral+vector+results+in+phenotypic+correction+of+canine+hemophilia+B+without+liver+toxicity+or+thrombocytopenia.">A gene-deleted adenoviral vector results in phenotypic correction of canine hemophilia B without liver toxicity or thrombocytopenia.</a> Blood. 102 (7), 2403-2411 (2003).</li><li>Cots, D., Bosch, A., Chillon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helper+dependent+adenovirus+vectors:+progress+and+future+prospects.">Helper dependent adenovirus vectors: progress and future prospects.</a> Curr Gene Ther. 13 (5), 370-381 (2013).</li><li>Mizuguchi, H., Kay, M. 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The protocol presented here allows purification of HCAdV vectors based on human adenovirus type 5 based on previously described procedures 4,12. The HCAdV genome within the pAdFTC plasmid is devoid of all adenovirus genes and only carries the 5&#39;- and 3&#39;- ITRs and the packaging signal. In this strategy the HV AdNG163R-2 4 provides all necessary genes for efficient virus production in trans. This offers a packaging capacity of up to 35 kb, which clearly outcompetes first and second ge...

For gene therapeutic applications it is of great importance to avoid cytotoxic and immunogenic side effects caused by expression of viral proteins, the transgene itself, or by incoming viral proteins. Adenovirus vectors (AdV) are widely used to introduce foreign DNA into a wide variety of cells to investigate the impact of transgene expression 1,2. The most advanced version of AdV is represented by high-capacity adenovirus vectors (HCAdV) lacking all viral coding sequences 3,4 and thereby offering a packaging capacity up to 35 kb combined with low immunogenicity and low toxicity 5-8. Due to their high packaging capacity they allow delivery of large or multiple transgenes using a single vector dose. Therefore, they represent a valuable tool for the research community.
In contrast to first- or second-generation AdV lacking the early genes E1 and/or E3 that can be easily produced using commercial kits, vector genome construction and virus production of HCAdV is more complex. The system for the construction of HCAdV genomes is based on the plasmid pAdFTC carrying a HCAdV genome devoid of all viral coding sequences and the shuttle plasmid pHM5 9-12. Any gene of interest of up to 14 kilobases (kb) can be cloned into the shuttle vector pHM5 in which the multiple cloning site is flanked by recognition/cleaving sites of the homing endonucleases PI-SceI and I-CeuI. Therefore, a cloned gene of interest can be released by consecutive PI-SceI and I-CeuI digests for subsequent directed insertion into the same restriction sites present in the HCAdV genome contained in the plasmid pAdFTC. In pAdFTC the transgene insertion site located between the PI-SceI and I-CeuI cleavage sites is flanked by stuffer DNA and the noncoding adenoviral sequences required for genome packaging such as the 5&#39; and 3&#39; inverted terminal repeats (ITRs) at both ends and the packaging signal downstream of the 5&#39;ITR. The additional stuffer DNA provides optimal size of the final HCAdV genome ranging from 27 to 36 kb to ensure efficient packaging during virus production. Since pAdFTC is a large plasmid with up to 45 kb (dependent on the size of the inserted transgene) and the usage of homing endonucleases with comparably long DNA recognition sites exhibits strong DNA binding, several cleanup steps are necessary during transfer of the transgene from pHM5 to pAdFTC. Careful handling avoiding shearing forces is recommended.
The ITRs of the HCAdV genome are flanked by NotI restriction enzyme recognition sites located directly upstream of the 5&#39;ITR and downstream of the 3&#39;ITR 12. Therefore, HCAdV can be released by NotI digest for subsequent transfection of the viral genome into the HCAdV producer cell line. Note that the usage of the restriction enzyme NotI for release of the viral genome from the plasmid pAdFTC implies that the inserted transgene is devoid of NotI DNA recognition sites. The HEK293 cell based producer cells (116 cells) stably express Cre recombinase. For virus amplification 116 cells are co-infected with a helper virus (HV) providing all AdV genes needed for replication and packaging in trans 3,4. The HV is a first-generation AdV with a floxed packing signal which is removed during virus amplification by Cre recombinase expressed in 116 cells 4. This ensures that predominantly HCAdV genomes containing an intact packaging signal are encapsidated.
Pre-amplification of the HCAdV is performed by conducting serial passaging steps in 116 cells grown on surfaces in tissue culture dishes. After each passage viral particles are released from infected cells by conducting three consecutive freeze-thaw steps. With every passage increasing numbers of cells are infected with 1/3 of cell lysate from the preceding passage. Finally lysate from the last pre-amplification step is used to infect producer cells grown in suspension in a spinner flask for large scale amplification. Virions are purified from the suspension cells by performing ultracentrifugation in a cesium chloride density gradient 4,12. With this procedure empty particles and fully assembled particles are separated into two distinct bands. To further concentrate the HCAdV particles a second non-gradual ultracentrifugation step is performed. Subsequently the resulting band containing the HCAdV is collected and dialyzed against a physiological buffer. Final vector preparations are characterized with respect to numbers of absolute viral particles, infectious particles and HV contamination levels. Absolute viral particles can be determined by lysing viral particles and measuring the absorbance at 260 nm or by performing quantitative real-time PCR (qPCR) 12. Infectivity of the purified virus particles can be determined by qPCR measuring HCAdV genomes present within infected cells 3 hr post-infection.

<table border="0" cellpadding="0" cellspacing="0" width="896">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:312px;">I-CeuI</td>
			<td style="width:147px;">New England Biolabs</td>
			<td style="width:123px;">R0699S</td>
			<td style="width:315px;">restriction digest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">PI-SceI</td>
			<td style="width:147px;">New England Biolabs</td>
			<td>R0696S</td>
			<td>restriction digest</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">T4 Ligase</td>
			<td style="width:147px;">New Engand Biolabs</td>
			<td>M0202S</td>
			<td>ligation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">SwaI</td>
			<td style="width:147px;">New England Biolabs</td>
			<td>R0604S</td>
			<td>restriction digest</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:312px;">NotI</td>
			<td style="width:147px;">New England Biolabs</td>
			<td>R0189S</td>
			<td>restriction digest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Calf Intestinal Alkaline Phosphatase (CIP)</td>
			<td style="width:147px;">New England Biolabs</td>
			<td>M0290S</td>
			<td>dephosphorylation of digested plasmids</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Hygromycin B</td>
			<td style="width:147px;">PAN Biotech</td>
			<td style="width:123px;">P02-015</td>
			<td>selection of CRE expressing 116 cells</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">DMEM</td>
			<td style="width:147px;">PAN Biotech</td>
			<td>P04-03590&#160;&#160;&#160;</td>
			<td>Hek293T cell culture medium</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">Minimal Essential Medium (MEM) Eagle</td>
			<td style="width:147px;">PAN Biotech</td>
			<td style="width:123px;">P04-08500</td>
			<td>116 cell culture medium&#160;&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Dulbecco&#8217;s phosphate buffer saline (DPBS)</td>
			<td style="width:147px;">PAN Biotech</td>
			<td style="width:123px;">P04-36500</td>
			<td>washing of cells, resuspension of cells</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:312px;">250 ml storage bottle</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:123px;">CLS430281-24EA</td>
			<td>infection of 116 cells grown in suspension</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">500mL PP CentrifugeTubes</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:123px;">CLS431123-36EA</td>
			<td>sedimentation of cells from suspension culture</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">Spinner flask</td>
			<td style="width:147px;">Bellco</td>
			<td style="width:123px;">1965-61030</td>
			<td>growth of 116 cells in suspension</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">Ultra Clear Ultracentrifuge tubes</td>
			<td style="width:147px;">Beckmann Coulter</td>
			<td style="width:123px;">344059</td>
			<td>density gradient centrifugation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">Ultracentrifuge</td>
			<td style="width:147px;">Beckmann Coulter</td>
			<td style="width:123px;">&#160;--</td>
			<td>density gradient centrifugation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">SW-41 rotor</td>
			<td style="width:147px;">Beckmann Coulter</td>
			<td style="width:123px;">&#160;--</td>
			<td>density gradient centrifugation</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:312px;">Spectrum Laboratories Spectrapor Membrane</td>
			<td style="width:147px;">VWR</td>
			<td style="width:123px;">132129</td>
			<td>dialysis tubing</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">ready-to-use dialysis cassettes&#160;</td>
			<td style="width:147px;">Thermo</td>
			<td style="width:123px;">66383</td>
			<td>dialysis</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">one shot DH10B electrocompetent E. coli</td>
			<td style="width:147px;">invitrogen</td>
			<td>C4040-52</td>
			<td>transformation of ligation reactions</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">PureYield&#8482; Plasmid Midiprep System</td>
			<td>Promega</td>
			<td>A2495</td>
			<td>midiprep</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">peqGOLD Tissue DNA Mini Kit&#160;</td>
			<td style="width:147px;">Peqlab</td>
			<td>12-3396-02</td>
			<td>isolation of genomic DNA</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">SuperFect Transfection Reagent</td>
			<td style="width:147px;">Qiagen</td>
			<td style="width:123px;">301305</td>
			<td>tranfection of 116 cells</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">opti MEM (10 % FBS)&#160;</td>
			<td style="width:147px;">Gibco</td>
			<td>31985-062</td>
			<td>transfection of 116 cells</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">iQ SYBR Green Supermix</td>
			<td>BioRad</td>
			<td>&#160;170-8882</td>
			<td>q-PCR</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">CFX 96 C1000 touch&#160;</td>
			<td style="width:147px;">Biorad</td>
			<td style="width:123px;"></td>
			<td>qPCR machine</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">Phenol/Chloroform/Isoamyl alcohol&#160;</td>
			<td style="width:147px;">Carl Roth</td>
			<td style="width:123px;">A156.1</td>
			<td>purification of DNA</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">Cesium chloride</td>
			<td style="width:147px;">Carl Roth</td>
			<td style="width:123px;">8627.1</td>
			<td>density gradient centrifugation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">sodium acetate 99%</td>
			<td style="width:147px;">Carl Roth</td>
			<td style="width:123px;">6773.2</td>
			<td>DNA precipitation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">LB medium&#160;</td>
			<td style="width:147px;">Carl Roth</td>
			<td style="width:123px;">X968.3</td>
			<td>bakterial growth medium</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">ethanol&#160; 99,8% pure</td>
			<td style="width:147px;">Carl Roth</td>
			<td>9065.5&#160;</td>
			<td>DNA precipitation and washing</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">SDS 99,5%</td>
			<td style="width:147px;">Carl Roth</td>
			<td>2326.2</td>
			<td>lysis&#160; buffer</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">EDTA</td>
			<td style="width:147px;">Carl Roth</td>
			<td>8043.2</td>
			<td>lysis&#160; buffer</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Tris-HCl 99%</td>
			<td style="width:147px;">Carl Roth</td>
			<td style="width:123px;">9090.3</td>
			<td>dialysis buffer/ lysis&#160; buffer</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">glycerol 99,5%</td>
			<td style="width:147px;">Carl Roth</td>
			<td style="width:123px;">3783.1</td>
			<td>dialysis buffer</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MgCl2 98,5%</td>
			<td style="width:147px;">Carl Roth</td>
			<td style="width:123px;">KK36.2</td>
			<td>dialysis buffer</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">NaCl</td>
			<td style="width:147px;">Carl Roth</td>
			<td style="width:123px;">3957.1</td>
			<td>optional dialysis buffer</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:312px;">KH2PO4</td>
			<td style="width:147px;">Carl Roth</td>
			<td style="width:123px;">3904.2</td>
			<td>optional dialysis buffer</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:312px;">sucrose</td>
			<td style="width:147px;">Carl Roth</td>
			<td style="width:123px;">9286.1</td>
			<td>optional dialysis buffer</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:312px;">Na2HPO4x2H2O</td>
			<td style="width:147px;">Carl Roth</td>
			<td>4984.2</td>
			<td>optional dialysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">1,5ml tubes</td>
			<td style="width:147px;">sarstedt</td>
			<td style="width:123px;">72,730,005</td>
			<td>storage of&#160; virus preparations at -80&#176;C</td>
		</tr>
	</tbody>
</table>

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.

Here representative examples for cloning, amplification and purification of HCAdV preparations are shown. An overview of the cloning strategy (Figure 1) and representative examples for cloning and release of the HCAdV genome by restriction enzyme digest are provided (Figure 2). A typical restriction pattern after release of the GOI-expression cassette from pHM5 by PI-SceI and I-CeuI digest and subsequent phenol-chloroform extraction and ...

Watch this Scientific Journal Video about Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5 at JoVE.com

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

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 ...

The overall goal of this procedure is to produce a gene-deleted, high-capacity adenoviral vector for delivery and expression of therapeutic transgenes in living cells or organisms. This method can help the gene therapy field to deliver large trans genes that do not fit into other viral vectors. The main advantage of this technique is that high-capacity adenoviruses are devoid of viral genes.Using these vectors up to 35 kb of foreign DNA can be delivered. The application of this technique extends to our therapy of inherited diseases because it helps to deliver therapeutic ventures for gene correction or gene addition. This method provides an advanced tool for the biodelivery of therapeutic transgenes and can be applied in cell culture as well as living organisms.Visual demonstration of this method is crucial as vector amplification and vector purification requires some experience. One needs to have right touch during vector purification. Check an aliquot of one in ten diluted linearized high-capacity adenovirus genome expression plasmid by gel electrophresis.A nine kilobase fragment for the pAd-FTC plasmid backbone and a second, 28 to 36 kilobase DNA fragment for the high-capacity adenovirus genome, plus gene of interest, should be detectable. Next, transfect a 60 milometer dish of one one six cells at 50 to 80%confluency with five micrograms of the linearized high-capacity adenovirus genome, according to standard procedures. 16 to 18 hours post-transfection, carefully remove the medium and add three milliliters of fresh medium.Then, infect the cells with helper virus AdNG 163R2, applying five transducing units per cell. Incubate in a tissue culture incubator at 37 degrees Celsius and 5%CO2. And agitate the plate every 20 minutes during the first hour of incubation to equally distribute the helper virus to all cells.48 hours post infection, harvest the cells by flushing them from the culture dish using the culture medium. Reserve a 0.5 milliliter aliquot of the passage zero cells for isolating genomic DNA. Then, repeatedly freeze/thaw a 2.5 milliliter aliquot of passage zero cells by immersing in liquid nitrogen.Or placing the cells in the negative 80 degrees Celsius freezer and then transferring to a 37 degrees Celsius water bath 3 to 4 times. Mix the 2.5 milliliters of lysate with one milliliter of fresh medium and add helper virus at two transforming units per cell. Aspirate the media from a 60 millimeter dish of one one six cells at 90 to 95%confluency and add the viral mixture to the cells.Incubate the cells for 48 hours as before. Then, harvest the cells and repeat the freeze/thaw and infection procedures twice to obtain lysates of passage one and passage two. After the passage two lysate has been obtained, mix the 2.5 milliliters of lysate with 17.5 milliliters of fresh media and add two transforming units per cell of helper virus.Aspirate the media from an 80 to 100%confluent 150 millimeter dish of one one six cells and infect the cells with the viral mixture. 48 hours post infection, harvest the passage three lysate using the procedure just shown. To begin this step, add 900 milliliters of pre-warmed media to a three liter spinner culture flask.Remove the media from at least 10 individual 150 millimeter dishes of one one six cells at 90 to 100%confluency. And flush off the cells with 10 milliliters of fresh, pre-warmed media. Pipette up and down several times to get a homogeneous cell suspension.And then transfer the cells directly into the spinner flask. Incubate the spinner flask on a magnetic stirrer in a tissue culture incubator for 24 hours at 37 degrees Celsius and 5%CO2. Adjust the magnetic stirrer to 70 RPM to avoid attachment of cells to the glass surfaces.24 hours after setting up the spinner culture flask, add 500 milliliters of fresh media. Repeat this step again after a further 24 hours. 72 hours after setting up the culture, add one liter of fresh media to the flask, resulting in a total volume of three liters of cell suspension.24 hours later, harvest the one one six suspension cells by centrifugation for ten minutes at 500 times g at room temperature. Discard the supernatant and resuspend the pellet in 150 milliliters of media by pipetting up and down about eight to ten times. Transfer the cells to a 250 milliliter storage bottle equipped with a sterile magnetic stir bar.Then, co-infect the cells with the passage three lysate and two transforming units of helper virus per cell. Here the total cell number is about nine times ten to the eighth cells, therefore, one point eight times ten to the ninth transforming units are added. Stir the cell virus mixture on a magnetic stirrer in the tissue culture incubator for two hours at 60 RPM.Make sure that the storage bottle is not completely closed to allow air circulation. Two hours post infection, transfer the total volume of the cell virus mixture back into the three liter spinner culture flask. Add 1850 milliliters of fresh, pre-warmed media and incubate in the tissue culture incubator for 48 hours at 70 RPM.After 48 hours, transfer the cell virus suspension to 500 milliliter centrifuge tubes. And then, centrifuge for 10 minutes at 890 times g at room temperature. Remove the medium and resuspend the pelleted cells in a total volume of 28 milliliters of DPBS.Store the cell virus suspension at negative 80 degrees Celsius until ready for virus purification. To prepare the viral lysate for cesium chloride gradient purification, freeze/thaw the cell virus suspension four times. Then, centrifuge the viral lysate at 500 times g for eight minutes at room temperature.Then, collect the supernatant containing the high-capacity adenovirus. Carefully and slowly pipette cesium chloride solutions into the tubes in the following order, 0.5 milliliters of 1.5 grams per cubic centimeter solution, three milliliters of 1.35 grams per cubic centimeter solution and 3.5 milliliters of 1.25 grams per cubic centimeter solution. Overlay approximately 4.5 milliliters of cleared vector supernatant on top of the 1.25 grams per cubic centimeter layer.Centrifuge the gradients in an ultra centrifuge using a swing out rotor at 12 degrees Celsius for at least two hours at 226, 000 times g to separate the high-capacity adenovirus genome containing viral particles from empty particles and cell debris. Following the centrifugation, a diffuse band of cell debris forms on top of the tubes. Below this, two white bands can be observed.The upper band contains empty particles, whereas the lower band is the high-capacity adenovirus. Carefully remove the layers of cell debris and empty particles. Then, collect one milliliter of the lower bands from each tube.Transfer the virus into a sterile 50 milliliter tube. Add up to 24 milliliters of 1.35 grams per cubic centimeter cesium chloride solution to the collected virus particles and mix carefully. Fill the centrifuge tubes to the top with 1.35 grams per cubic centimeter cesium chloride virus solution.Then centrifuge overnight at 226, 000 times g at 12 degrees Celsius in an ultra centrifuge using a swing out rotor with slow acceleration and deceleration. Remove the upper layers from the top using a pipette. Then, collect the high-capacity adenovirus present in the prominent lower band using a fresh pipette tip.Finally, dialyse the collected virus particles for buffer exchange. And then, titer the final preparation according to the detailed instructions in the written protocol. This histogram shows absolute numbers of particles for a single vector preparation as determined with three different methods, OD titer measured by optical density, infections titer and helper virus contamination measured by qPCR.The ratio between the total infectious, high-capacity adenovirus particles and helper virus contamination levels measured by qPCR is shown here. If a fluorescent marker gene is present in your vector, fluorescent microscopy can be performed to monitor the pre-amplification of the vector and to characterize the infectivity of the final vector preparation. After watching this video, you should have a good understanding of how to produce gene deleted, high-capacity adenoviruses.Especially how to insert your transgene and how to amplify and purify the vectors.

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