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>
	<li>Benihoud, K., Yeh, P., Perricaudet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+vectors+for+gene+delivery.">Adenovirus vectors for gene delivery.</a> Curr Opin Biotechnol. 10 (5), 440-447 (1999).</li><li>Crystal, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus:+the+first+effective+in+vivo+gene+delivery+vector.">Adenovirus: the first effective in vivo gene delivery vector.</a> Hum Gene Ther. 25 (1), 3-11 (2014).</li><li>Parks, R. 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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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+adenoviral+helper-dependent+vector+results+in+long-term+therapeutic+levels+of+human+coagulation+factor+IX+at+low+doses+in+vivo.">A new adenoviral helper-dependent vector results in long-term therapeutic levels of human coagulation factor IX at low doses in vivo.</a> Blood. 99 (11), 3923-3930 (2002).</li><li>Jager, L., 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+rapid+protocol+for+construction+and+production+of+high-capacity+adenoviral+vectors.">A rapid protocol for construction and production of high-capacity adenoviral vectors.</a> Nat Protoc. 4 (4), 547-564 (2009).</li><li>Sambrook, J., Russell, D. 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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 ...

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Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Cerebral Blood Flow-Based Resting State Functional Connectivity of the Human Brain using Optical Diffuse Correlation Spectroscopy

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a ...

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 ...

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process ...

Microtubule Plus-End Dynamics Visualization in Huntington's Disease Model based on Human Primary Skin Fibroblasts

Transfection with a fluorescently labeled marker protein of interest in combination with time-lapse video microscopy is a classic method of studying the ...