NOTE: In the following, a protocol for geneti-chemical PEGylation of an Ad vector is described to detail. To enable specific coupling of the PEG moiety, an Ad5 vector was beforehand genetically modified by introducing a cysteine residue into the hexon protein at the hypervariable loop 5 as described in a previous publication36, and a maleimide-activated PEG compound is used as coupling compound.
1. Preparation of Buffers for Vector Purification by CsCL Step Gradients
<ol>
	<li>Prepare 400 mL of Adenovirus buffer (Ad-buffer) by adding 50 mM HEPES (4.76 g/400 mL) and 150 mM NaCl (3.504 g/400 mL) to double-distilled water (ddH2O). 350 mL of this buffer is needed to prepare the following three Ad-buffer variants.
	<ol>
		<li>Variant A: adjust 150 mL of Ad-buffer to pH 7.6 with NaOH.</li>
		<li>Variant B: add a final concentration of 10 mM TCEP [tris(2-carboxyethyl)phosphine), 0.143 g] to 50 mL and adjust it to pH 7.2 with HCl.</li>
		<li>Variant C: add a final concentration of 0.5 mM TCEP (0.022 g) to 150 mL and adjust it to pH 7.2 with HCl.</li>
		<li>Prepare the buffers in ddH2O and analytical grade chemicals. Use 100 mL each of variant A and variant C to prepare the CsCl buffers (see steps 1.2 and 1.3; 50 mL of each) necessary for the CsCl step gradients (see steps 3.2.6. and 3.2.18).</li>
	</ol>
	</li>
	<li>Preparing the CsCl step gradient buffer (&#961;CsCl = 1.41 g/cm3) in Ad-buffer 
	NOTE: This buffer will be needed in two different variations:
	<ol>
		<li>Weigh two aliquots of exactly 27.42 g of CsCl into 50 mL tubes.</li>
		<li>&#961;CsCl = 1.41/TCEP buffer: resolve CsCl in 40 mL of variant C of Ad-buffer.</li>
		<li>&#961;CsCl = 1.41 buffer: resolve CsCl in 40 mL of variant A of Ad-buffer.</li>
		<li>Check, and if necessary, adjust the pH with HCl. Fill up to exactly 50 mL with the corresponding Ad-buffer variant. To achieve the best separation results, the exact CsCl concentration and pH are crucial.</li>
		<li>Check the density (CsCl content) by weighing 1 mL (1.41 g) of each gradient buffer.</li>
	</ol>
	</li>
	<li>Preparing the CsCl step gradient buffer (&#961;CsCl = 1.27 g/cm3) in Ad-buffer 
	NOTE: This buffer is needed in two different variations:
	<ol>
		<li>Weigh two aliquots of exactly 18.46 g of CsCl in 50 mL tubes.</li>
		<li>&#961;CsCl = 1.27/TCEP buffer: resolve CsCl in 40 mL of variant C of Ad-buffer.</li>
		<li>&#961;CsCl = 1.27 buffer: resolve CsCl in 40 mL of variant A of Ad-buffer.</li>
		<li>Check, and if necessary, adjust the pH with HCl. Finally, fill up to exactly 50 mL with the corresponding Ad-buffer variant. To achieve the best separation results, the exact CsCl concentration and pH are crucial.</li>
		<li>Check the density (CsCl content) by weighing 1 mL (1.27 g) of each gradient buffer.</li>
	</ol>
	</li>
	<li>Sterilize all buffers (Ad-buffers and CsCl step gradient buffers) by filtration (using 0.45 &#181;m mesh pore size) into sterile bottles.</li>
	<li>After sterilization, to prevent oxidation of TCEP, saturate and overlay the buffers containing TCEP with argon by sparging the buffers with argon gas for about 30 s. Take care to only use sterile devices (e.g., sterile pipette tips) when applying the argon gas, and use bottles large enough to allow argon blubber. 
	NOTE: All buffers should be prepared fresh and protected from light and can be stored at 4 &#176;C for several days (especially the CsCl step gradient buffers that should only be kept for a few days).</li>
</ol>
2. Coupling Moieties: Storage and Preparation
NOTE: Moeities used for coupling to cysteines need to bear thiol-reactive groups. Maleimide-activated compounds will form stable thioether bonds with the genetically introduced cysteines. Alternatively, ortho-pyridyldisulfide (OPSS)-activated compounds can be used, which form bioresponsive disulfide bridges between the vector particles and coupling moiety. Lyophilized malPEG-750 as well as most other coupling reagents are sensitive to hydrolysis and should be stored dry in the form of lyophilized powders at -80 &#176;C.
<ol>
	<li>To prevent the build-up of condensing water upon thawing of the vials, store the vials in larger tubes (e.g., 50 mL tubes) filled with a cushion of silica gel beads. Store these tubes in an adequate larger container also filled with a silica gel bead cushion.</li>
	<li>Before tightly closing each tube and container, exchange air with argon gas. This can be achieved by placing the open tubes and containers into a desiccator, followed by removing and replacing the air with argon gas. Since argon gas is heavier than air, tubes and containers are filled up with argon gas and can now be placed into each other, with each lid tightly closed.</li>
	<li>Store containers at -80 &#176;C.</li>
	<li>When thawing, place the containers onto silica beads in a desiccator and slowly warm them up to room temperature, then open the container.</li>
	<li>When performing a coupling reaction, freshly resolve the amount of coupling substrate needed into the appropriate solvent. Take care not to add more than 10% of coupling solution to the final coupling reaction, especially if DMF or DMSO is used as the solvent for the coupling substrate.</li>
</ol>
3. Amplification, Purification and Chemical Modification of Ad Vectors:
<ol>
	<li>Amplification of Ad vectors 
	NOTE: The following steps must be performed using a safety cabinet according to local biological safety rules.
	<ol>
		<li>Transfect a replication deficient &#8710;E1 Ad vector containing one (or several) genetically introduced cysteine residue(s) into HEK 293 cells as described by Kratzer and Kreppel38.</li>
		<li>According to the protocol38, amplify the vector by sequential reinfections up to a final preparative infection of 15-20 large (15 cm) cell culture plates (approximately 1-2 x 107 cells/plate). 
		NOTE: Optimally, the last reinfection should be timed so that cells show full cytopathic effect (CPE) on the morning of purification and modification performance (see Figure 2).</li>
		<li>After the final amplification step, cells resuspended in Ad buffer + 10 mM TCEP (variant B) can be frozen at -80 &#176;C; however, for optimal yield of vector particles, an immediate follow-up of cell lysis and vector purification and modification is recommended.</li>
	</ol>
	</li>
	<li>Purification and chemical modification of Ad vectors 
	NOTE: Purification of vectors is performed according to the adenovirus purification protocol described in38 but under non-oxidizing conditions. Cell harvesting and lysis as well as vector purification and chemical modification can be performed within one day. Harvest and lyse the infected cells according to the protocol described previously38.
	<ol>
		<li>Collect cells by scraping the plates and transferring the supernatant to 200-500 mL centrifugation tubes.</li>
		<li>Spin the cell solution for 10 min at 400 x g.</li>
		<li>Resuspend the cell pellet in 4 mL of Ad-buffer + 10 mM TCEP (pH 7.2) (variant B) and transfer to a sterile 50 mL tube. If cell yield is low or only a weak CPE was induced, resuspend it in 2 mL.</li>
		<li>Rescue the vector by 3 cycles of freezing (in liquid nitrogen) and thawing (in a 37 &#176;C water bath).</li>
		<li>Spin for 10 min at 5,000 x g at 4 &#176;C.</li>
		<li>While centrifuging, prepare two equal CsCl step gradients:
		<ol>
			<li>First, as the lower phase, add 3 mL of 1.41 g/cm3 &#961;CsCl in Ad-buffer + 0.5 mM TCEP (pH 7.2, variant C) into the ultracentrifuge tubes. Mark the level of the lower phase on the tube before loading the upper phase.</li>
			<li>Carefully stack the upper phase of 5 mL of 1.27 g/cm3 &#961;CsCl in Ad-buffer + 0.5 mM TCEP (pH 7.2, variant C) by very slowly pipetting the 5 mL volume along the walls of the tube onto the lower phase. 
			NOTE: Since during coupling a high concentration of TCEP could react with the maleimide residue of malPEG-750 and lead to reduced coupling efficiency, purification by CsCl step gradient needs to be already performed under a reduced TCEP concentration.</li>
		</ol>
		</li>
		<li>Load the supernatant onto the CsCl gradient [either equally divide the supernatant onto both gradients or, in the case of a low vector yield (see above), load the supernatant onto only one column], and fill both gradients equally to the top with Ad buffer + 10 mM TCEP (pH 7.2, variant B).</li>
		<li>Adjust the weight of the opposite tubes to a 0.0 g weight difference.</li>
		<li>Ultracentrifuge for 2 h at 176,000 x g at 4 &#176;C.</li>
		<li>With a clamp, fix the ultracentrifugation tube to a stand, leaving the area around the lower phase level mark accessible. Place the gooseneck lamp above the tube. Around the mark, at the border between the lower and upper phases, a distinct band (the vector virions) should be visible (Figure&#160;3A-3C).</li>
		<li>Collect the vectors by puncturing the tube with a needle and aspiring the vector band into a syringe. Transfer collected vector virions to a 15 mL tube. 
		NOTE: Weaker bands above the Ad vector band contain incomplete vector particles and should be avoided for aspiration. The cell debris and green fluorescent protein (EGFP) of EGFP-expressing Ad-vectors will collect in the upper part of the ultracentrifuge tube (see Figure&#160;3A and 3B). This and the following steps up to coupling (step 3.2.16) should be performed speedily, as the vectors are now sensible to disulfide bonding due to the low TCEP concentration.</li>
		<li>To calculate the amount of coupling substrate needed for efficient complete binding of the substrate to all cysteine residues in the vector preparation, measure OD260:
		<ol>
			<li>According to the protocol described by Kratzer and Kreppel38, vortex a mixture of 10 &#181;L of the collected vector virions, 89 &#181;L of Ad buffer, and 1 &#181;L of 10% SDS. As a blank probe, vortex 99 &#181;L of Ad-buffer and 1 &#181;L of 10% SDS.</li>
			<li>To denature the virions, incubate both at 56 &#176;C for 10 min.</li>
			<li>Determine OD260.</li>
			<li>Calculate the physical vector titer per &#181;L using the following equation: OD260 x Factor of dilution x Empirically determined extinction coefficient of Ad (1.1 x 109 vector particles = 1 OD260 unit/&#181;L). Therefore, a measured OD260 of 1.36 of a vector diluted 1:10, would calculate to: 1.36 x 10 x 1.1 x 109 = approximately 1.5 x 1010 vector particles/&#181;L. 
			NOTE: This titer is only a rough estimation; however, it is accurate enough for the stoichiometric calculations outlined in the following.</li>
		</ol>
		</li>
		<li>Depending on the protein modified by introduction of a cysteine, determine the amount of coupling substrate (in grams) for an efficient coupling reaction, according to the following general equation: Virus titer x Volume x Number of cysteines x Excess of moiety x Molecular weight of moiety / 6,022 x 1023 = grams of coupling substrate. 
		NOTE: In this equation: 1) virus titer is the titer/&#181;L, determined by OD260 as described above, 2) volume accounts for the volume in &#181;L of aspired vector used in the coupling reaction, 3) the number of cysteines is the number of proteins into which the cysteine residue was introduced per vector particle, 4) excess of moiety is the factor of moiety excess necessary for an efficient coupling reaction (50x), and 5) the molecular weight of moiety must be introduced as Da (not kDa).</li>
		<li>Freshly resolve the amount of compound (or feasible amount) needed in the appropriate solvent. 
		NOTE: As the amount of coupling substrate needed is low and difficult to weigh but expensive, weigh the minimal amount of compound possible and dissolve it in an appropriate concentration for application to the coupling reaction.</li>
		<li>Transfer the vector solution to an appropriate size tube (e.g., 2 mL tube) and add the calculated amount of coupling compound stock solution to the vector.</li>
		<li>Gently mix the solution by overhead rotation for 1 h at room temperature.</li>
		<li>Fill up the vector solution to 6 mL total volume with Ad-buffer (pH 7.6, variant A). 
		NOTE: This step must be performed before the solution is applied to the step gradient to ensure that the vector solution, which still contains CsCl from the first step gradient, has a density below 1.27 g/mL.</li>
		<li>In a second CsCl banding step, purify the vector from the unreacted coupling moiety:
		<ol>
			<li>Prepare two equal CsCl step gradients by performing the following steps for each: 1) Lower phase: 3 mL of 1.41 g/cm3 &#961;CsCl in Ad-buffer (pH 7.6, variant A), 2) mark level of lower phase on the tube before loading upper phase, and 3) upper phase: 5 mL of 1.27 g/cm3 &#961;CsCl in Ad-buffer (pH 7.6, variant A). 
			NOTE: After coupling has taken place, buffers without TCEP are used, as no free thiol groups should now be present on the vector capsids.</li>
			<li>Load the supernatant onto the CsCl gradient and fill both gradients equally to the top with Ad-buffer (pH 7.6, variant A).</li>
			<li>Adjust the weights of the opposite tubes to a 0.0 g weight difference.</li>
		</ol>
		</li>
		<li>Ultracentrifuge for 2 h at 176,000 x g at 4 &#176;C.</li>
		<li>Shortly before the end of ultracentrifugation, equilibrate a PD-10 column 5 times with 5 mL of Ad-buffer (pH 7.6, variant A).</li>
		<li>As done in step 3.2.10, fix the ultracentrifugation tube to stand leaving area around the lower phase level mark accessible. Place the gooseneck lamp above the tube. Again, around the border between the lower and upper phases, a distinct band (the vector virions) should be visible (Figure 3C).</li>
		<li>Collect the vectors by puncturing the tube with a needle and aspiring the vector band into a syringe, and transfer the vector to a 15 mL tube. Take care to collect the virions of both gradient tubes together as a 2.5 mL total volume or less. If a smaller volume is collected, fill to obtain a 2.5 mL total volume with Ad buffer (variant A).</li>
		<li>Load the 2.5 mL onto the equilibrated PD-column. Discard the flow-through.</li>
		<li>Add 3 mL of Ad buffer (variant A) onto the column and collect the flow-through (containing the purified vector virions).</li>
		<li>Add glycerol (333 &#181;L) to obtain a final concentration of 10% and divide into suitable aliquots (e.g., 400 &#181;L each), and include an aliquot of 20 &#181;L for titer determination by OD260 measurement.</li>
		<li>Store the vector solution at -80 &#176;C.</li>
		<li>Determine the titer of vector by measuring the OD260 of 20 &#181;L vector solution, 79 &#181;L of Ad-buffer (variant A), and 1 &#181;L of 10% SDS as described in step 3.2.12. As a blank, use 79 &#181;L of Ad-buffer (variant A), 10 &#181;L of 10% glycerol, and 1 &#181;L of 10% SDS.</li>
		<li>Calculate the vector titer as: OD260 x Factor of dilution x 1.1 x 109 vector particles/&#181;L. 
		As prepared in step 3.2.27, calculate: OD260 x 5 x 1.1 x 109 vector particles/&#181;L</li>
	</ol>
	</li>
</ol>
4. Verification of Coupling by SDS-PAGE:
NOTE: If compounds with sufficiently high molecular weights are used for coupling to Ad virions, coupling can be verified by polyacrylamide gel electrophoresis (SDS-PAGE). Successful coupling should then result in a shift of the protein band corresponding to the modified Ad virion protein, compared to the protein in the unmodified Ad virion (see Figure 4).
<ol>
	<li>According to the calculated titer of vector (step 3.2.28), separate 1-2 x 1010 vector particles by SDS-PAGE (8%).</li>
	<li>Develop the gel by silver-staining of the gel39 to detect even weak protein bands:
	<ol>
		<li>Prepare buffers for silver staining (the amounts needed for 100 mL of buffer are indicated in brackets):
		<ol>
			<li>Prepare buffer 1 by mixing 38 mL of ddH2O, 50% MeOH (50 mL of 100 MeOH), 12% AcOH (12 mL of 100% AcOH), and 0.0185% HCHO (50 &#181;L of 37% HCHO).</li>
			<li>Prepare buffer 2 by adding 50% EtOH (50 mL of 100% EtOH) to 50 mL of ddH2O.</li>
			<li>Prepare buffer 3 by adding 0.127 g/L Na2S2O3 (12.744 mg) to 100 mL of ddH2O.</li>
			<li>Prepare buffer 4 by adding 2 g/L AgNO3 (0.2 g) and 0.0185% HCHO (50 &#181;L of 37% HCHO) to 100 mL of ddH2O.</li>
			<li>Prepare buffer 5 by adding 60 g/L Na2CO3 (6 g), 0.0185% HCHO (50 &#181;L of 37% HCHO), and 2.5 mg/L Na2S2O3 (0.256 mg) to ddH2O to obtain a final volume of 100 mL.</li>
			<li>Prepare buffer 6 by mixing 38 mL of ddH2O, 50% MeOH (50 mL of 100% MeOH), and 12% AcOH (12 mL of 100% AcOH).</li>
			<li>Prepare buffer 7 by mixing 60 mL of ddH2O, 30% MeOH (30 mL of 100% MeOH), and 10% glycerine (10 mL of 100% glycerine).</li>
			<li>Prepare buffer 8 by mixing 10% glycerine (10 mL of 100% glycerine) with 90 mL of ddH2O.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Performing silver staining
	<ol>
		<li>Fix the gel in buffer 1 for 30 min.</li>
		<li>Wash the gel in buffer 2 for 15 min.</li>
		<li>Pretreat the gel in buffer 3 for 1 min.</li>
		<li>Wash the gel 3 times in ddH2O for 20 s.</li>
		<li>Impregnate the gel in buffer 4 for 20 min.</li>
		<li>Wash the gel 2 times in ddH2O for 20 s.</li>
		<li>Develop the gel in buffer 5 until bands are visible (10 s to 10 min).</li>
		<li>Wash the gel 2 times in ddH2O for 2 min.</li>
		<li>Stop development in buffer 6 for 5 min.</li>
		<li>Conserve the gel in buffer 7 for 20 min.</li>
		<li>Store the gel in buffer 8. 
		NOTE: Coupling efficiency can be determined by densitometric quantification of the modified and unmodified bands of the targeted capsomere.</li>
	</ol>
	</li>
</ol>

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C., 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=Human+erythrocytes+bind+and+inactivate+type+5+adenovirus+by+presenting+Coxsackie+virus-adenovirus+receptor+and+complement+receptor+1.">Human erythrocytes bind and inactivate type 5 adenovirus by presenting Coxsackie virus-adenovirus receptor and complement receptor 1.</a> Blood. 113, 1909-1918 (2009).</li><li>Waddington, S. 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=Adenovirus+serotype+5+hexon+mediates+liver+gene+transfer.">Adenovirus serotype 5 hexon mediates liver gene transfer.</a> Cell. 132, 397-409 (2008).</li><li>Kalyuzhniy, O., 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=Adenovirus+serotype+5+hexon+is+critical+for+virus+infection+of+hepatocytes+in+vivo.">Adenovirus serotype 5 hexon is critical for virus infection of hepatocytes in vivo.</a> Proceedings of the National Academy of Sciences USA. 105, 5483-5488 (2008).</li><li>Vigant, F., 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=Substitution+of+hexon+hypervariable+region+5+of+adenovirus+serotype+5+abrogates+blood+factor+binding+and+limits+gene+transfer+to+liver.+Molecular+Therapy.">Substitution of hexon hypervariable region 5 of adenovirus serotype 5 abrogates blood factor binding and limits gene transfer to liver. Molecular Therapy.</a> The Journal of the American Society of Gene Therapy. 16, 1474-1480 (2008).</li><li>Jonsson, M. I., 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=Coagulation+factors+IX+and+X+enhance+binding+and+infection+of+adenovirus+types+5+and+31+in+human+epithelial+cells.">Coagulation factors IX and X enhance binding and infection of adenovirus types 5 and 31 in human epithelial cells.</a> Journal of Virology. 83, 3816-3825 (2009).</li><li>Bradshaw, A. C., 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=Requirements+for+receptor+engagement+during+infection+by+adenovirus+complexed+with+blood+coagulation+factor+X.">Requirements for receptor engagement during infection by adenovirus complexed with blood coagulation factor X.</a> PLoS Pathogen. 6, e1001142 (2010).</li><li>Duffy, M. R., Bradshaw, A. C., Parker, A. L., McVey, J. H., Baker, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cluster+of+basic+amino+acids+in+the+factor+X+serine+protease+mediates+surface+attachment+of+adenovirus/FX+complexes.">A cluster of basic amino acids in the factor X serine protease mediates surface attachment of adenovirus/FX complexes.</a> Journal of Virology. 85, 10914-10919 (2011).</li><li>Xu, Z., 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=Coagulation+factor+X+shields+adenovirus+type+5+from+attack+by+natural+antibodies+and+complement.">Coagulation factor X shields adenovirus type 5 from attack by natural antibodies and complement.</a> Nature Medicine. 19, 452-457 (2013).</li><li>Prill, J. -. M., 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=Modifications+of+adenovirus+hexon+allow+for+either+hepatocyte+detargeting+or+targeting+with+potential+evasion+from+Kupffer+cells.+Molecular+Therapy.">Modifications of adenovirus hexon allow for either hepatocyte detargeting or targeting with potential evasion from Kupffer cells. Molecular Therapy.</a> The Journal of the American Society of Gene Therapy. 19, 83-92 (2011).</li><li>Zaiss, A. K., 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=Hepatocyte+Heparan+Sulfate+Is+Required+for+Adeno-Associated+Virus+2+but+Dispensable+for+Adenovirus+5+Liver+Transduction+In+Vivo.">Hepatocyte Heparan Sulfate Is Required for Adeno-Associated Virus 2 but Dispensable for Adenovirus 5 Liver Transduction In Vivo.</a> Journal of Virology. 90, 412-420 (2015).</li><li>Delgado, C., Francis, G. E., Fisher, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+uses+and+properties+of+PEG-linked+proteins.">The uses and properties of PEG-linked proteins.</a> Critical Reviews in Therapeutic Drug Carrier Systems. 9, 249-304 (1992).</li><li>Parveen, S., Sahoo, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomedicine:+clinical+applications+of+polyethylene+glycol+conjugated+proteins+and+drugs.">Nanomedicine: clinical applications of polyethylene glycol conjugated proteins and drugs.</a> Clin. Pharmacokinet. 45, 965-988 (2006).</li><li>O'Riordan, C. R., 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=PEGylation+of+adenovirus+with+retention+of+infectivity+and+protection+from+neutralizing+antibody+in+vitro+and+in+vivo.">PEGylation of adenovirus with retention of infectivity and protection from neutralizing antibody in vitro and in vivo.</a> Human Gene Therapy. 10, 1349-1358 (1999).</li><li>Subr, V., 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=Coating+of+adenovirus+type+5+with+polymers+containing+quaternary+amines+prevents+binding+to+blood+components.">Coating of adenovirus type 5 with polymers containing quaternary amines prevents binding to blood components.</a> Journal of Controlled Release. 135, 152-158 (2009).</li><li>Kreppel, F., Gackowski, J., Schmidt, E., Kochanek, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+genetic+and+chemical+capsid+modifications+enable+flexible+and+efficient+de-+and+retargeting+of+adenovirus+vectors.+Molecular+Therapy.">Combined genetic and chemical capsid modifications enable flexible and efficient de- and retargeting of adenovirus vectors. Molecular Therapy.</a> The Journal of the American Society of Gene Therapy. 12, 107-117 (2005).</li><li>Corjon, S., 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=Targeting+of+adenovirus+vectors+to+the+LRP+receptor+family+with+the+high-affinity+ligand+RAP+via+combined+genetic+and+chemical+modification+of+the+pIX+capsomere.">Targeting of adenovirus vectors to the LRP receptor family with the high-affinity ligand RAP via combined genetic and chemical modification of the pIX capsomere.</a> Molecular Therapy: The Journal of the American Society of Gene Therapy. 16 (11), 1813-1824 (2008).</li><li>Prill, J. -. M., 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=Modifications+of+adenovirus+hexon+allow+for+either+hepatocyte+detargeting+or+targeting+with+potential+evasion+from+Kupffer+cells.+Molecular+Therapy.">Modifications of adenovirus hexon allow for either hepatocyte detargeting or targeting with potential evasion from Kupffer cells. Molecular Therapy.</a> The Journal of the American Society of Gene Therapy. 19, 83-92 (2011).</li><li>Krutzke, 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=Substitution+of+blood+coagulation+factor+X-binding+to+Ad5+by+position-specific+PEGylation:+Preventing+vector+clearance+and+preserving+infectivity.">Substitution of blood coagulation factor X-binding to Ad5 by position-specific PEGylation: Preventing vector clearance and preserving infectivity.</a> Journal of Controlled Release. 235, 379-392 (2016).</li><li>Prill, J. -. M., 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=Traceless+bioresponsive+shielding+of+adenovirus+hexon+with+HPMA+copolymers+maintains+transduction+capacity+in+vitro+and+in+vivo.">Traceless bioresponsive shielding of adenovirus hexon with HPMA copolymers maintains transduction capacity in vitro and in vivo.</a> PloS One. 9, e82716 (2014).</li><li>Kratzer, R. F., Kreppel, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production,+Purification,+and+Titration+of+First-Generation+Adenovirus+Vectors.">Production, Purification, and Titration of First-Generation Adenovirus Vectors.</a> Methods in Molecular Biology. , 377-388 (2017).</li><li>Blum, H., Beier, H., Gross, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+silver+staining+of+plant+proteins,+RNA+and+DNA+in+polyacrylamide+gels.">Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels.</a> ELECTROPHORESIS. 8, 93-99 (1987).</li></ol>

The authors have nothing to disclose.

The efficiency by which the genetically introduced cysteines can be chemically modified is typically 80-99%, and certain variables influence this efficiency. First, it is paramount that the genetically introduced cysteines do not undergo premature oxidation. While being well-protected in the reducing environment of the producer cells, it is mandatory to provide a non-oxidative environment after releasing vector particles from the producer cells and during chemical modification. To this end, reducing reagents can be used ...

Adenoviruses (Ad), members of the family Adenoviridae, are non-enveloped DNA viruses of which more than 70 types so far have been identified (http://hadvwg.gmu.edu). Depending on hemaglutination properties, genome structure, and sequencing results, the 70 Ad types can be divided into seven species (human adenoviruses A to G)1,2. The human Ad genome is 38 kb in size and encapsulated by an icosahedral nucleocapsid3. Due to their abundance, the capsid protein hexon, penton base, and fiber are all referred to as major capsid proteins. The most abundant and largest capsid protein hexon forms 20 capsid facets, each consisting of 12 hexon homotrimers4,5. Penton, located on each icosahedral edge (vertex), consists of pentamers of a penton base and represents the base for the vertex spike that is built of glycosylated fiber trimers5,6. The native Ad cell entry is basically composed of two major steps. First, the fiber knob binds to the primary receptor. In Ad types from species A, C, E, and F, this is the coxsackie and adenovirus receptor (CAR). This interaction brings the virion into spatial proximity of the cell surface, thereby facilitating interactions between cellular integrins and RGD-motif in the penton base and consequently inducing cellular responses. Second, changes in the cytoskeleton lead to internalization of the virion and transport to the endosome7. Upon partial disassembly in the endosome, the virion is released to the cytoplasm und ultimately travels to the nucleus for replication.
While Ad can be delivered locally (e.g., for genetic vaccination), systemic delivery through the bloodstream as required for onco-virotherapy faces several barriers. While circulating in the bloodstream, injected virions encounter the defense system of the host&#39;s immune system, leading to fast neutralization of the virus-based vectors and rendering Ad-based vectors extremely inefficient in systemic applications. Furthermore, the natural hepatotropism of Ad interferes with systemic delivery and must be resolved to redirect Ad to its new target cells.
Germline-encoded natural IgM antibodies of the innate immune system rapidly recognize and bind highly repetitive structures on the surface of the virion8,9. These immune complexes then activate the classical and non-classical pathways of the complement system, leading to fast complement-mediated neutralization of a large portion of the virions8. A second pathway resulting in major removal of Ad virions is mediated by macrophages10&#160;and associated with acute toxic and haemodynamic side effects11,12. In the case of Ad in particular, Kupffer cells residing in the liver bind to and phagocytically take up the Ad virions via specific scavenger receptors, thereby eliminating them from the blood13,14,15. Specific scavenger receptors have also been identified on liver sinusoidal endothelial cells (LSE cells)16,and LSE cells also seem to contribute to vector elimination17, but to what extent still needs clarification. Furthermore, some Ad types and their&#160;derived vectors are efficiently sequestered by human erythrocytes18&#160;to which they bind via CAR or the complement receptor CR119.&#160;Of note, this sequestering mechanism cannot be studied in the mouse model system as in contrast to human erythrocytes, mouse erythrocytes do not express CAR.
Specific anti-Ad antibodies generated by the adaptive immune system after exposure to Ad either due to previous infections with Ad or after the first delivery in systemic applications raise a further barrier to effective use of Ad vectors, and they should be evaded in efficient systemic delivery.
Finally, the strong hepatotropism of some Ad types (including Ad5) severely hinders application of Ad in systemic therapy. This tropism resulting in hepatocyte transduction is due to the high affinity of the Ad virion to blood coagulation factor X (FX), mediated by the interaction of FX with the Ad hexon protein20,21,22. FX bridges the virion to heparin sulfate glycans (HSPGs) on the surface of hepatocytes20,23,24,25. A crucial factor for this interaction seems to be the specific extent of N- and O-sulfation of the HSPGs in liver cells24, which is distinct from HSPGs on other cell types. In addition to this FX-mediated pathway, recent studies suggest further pathways not yet identified that result in Ad transduction of hepatocytes26,27,28.
Recently, it has been shown that FX is not only involved in hepatocyte transduction of Ad, but also by binding, the virion shields the virus particle against neutralization by the complement system26. Reduction of hepatocyte transduction by preventing FX binding, therefore, would create the unwanted side effect of increasing Ad neutralization via the innate immune system.
A profound knowledge of the complex interactions between vectors and host organisms is therefore necessary to develop more efficient vectors for systemic applications that circumvent the obstacles imposed by the host&#39;s organism.
One strategy that has been originally used for therapeutic proteins has been adapted for Ad vectors to at least partially overcome the above described barriers. Antigenicity and immunogenicity of therapeutic protein compounds could be reduced by coupling to polyethylene glycol (PEG)29,30. Hence, the covalent coupling of polymers such as PEG or poly[N-(2-hydroxypropyl)methacrylamid] (pHPMA) to the capsid surface shields the vector from unwanted vector-host interactions. Commonly, polymer coupling targets &#949;-amine groups from lysine side residues that are randomly distributed on the capsid surface. Vector particles in solution are, due to the hydrophilic nature of the attached polymers, surrounded by a stable water shell that reduces the risk of immune cell recognition or enzymatic degradation. Moreover, PEGylated Ad vectors were shown to evade neutralization by anti-hexon antibodies in vitro and in pre-immunized mice in vivo31. In contrast to genetic capsid modifications, chemical coupling of polymers is performed after production and purification, allowing not only for the use of conventional producer cells and production of high titer vector stocks, but also for simultaneous modification of thousands of amino acids on the capsid surface. However, amine-directed shielding occurs randomly throughout the whole capsid surface, resulting in high heterogeneities and not allowing for modification of specific capsomers. Furthermore, the large polymer moieties required for beneficial effects impair virus bioactivity32.
To overcome these limitations, Kreppel et al.33 introduced a geneti-chemical concept for vector re- and de-targeting. Cysteines were genetically introduced into the virus capsid at solvent-exposed positions like fiber HI-loop33, protein IX34, and hexon35,36. Although not naturally-occurring, cysteine-bearing Ad vectors can be produced at high titers in normal producer cells. Importantly, insertion of cysteines in certain capsomers and in different positions within a single capsomer allows for highly specific modifications of thiol group-reactive moieties. This geneti-chemical approach has been shown to overcome numerous obstacles in Ad vector design. The combination of amine-based PEGylation for detargeting and thiol-based coupling of transferrin to the fiber knob HI-loop has been proven to successfully retarget modified Ad vectors to CAR-deficient cells33. Since hexon is involved in most undesired interactions (neutralizing antibodies, blood coagulation factor FX), thiol-based modification strategies were also applied to hexon. Coupling small PEG moieties to HVR5 of hexon prevented Ad vector particles to transduce SKOV-3 cells in the presence of FX, whereas large PEG moieties increased hepatocyte transduction14,35. Ad vector particles carrying mutations in the fiber knob inhibiting CAR binding and in HVR7 inhibiting binding of FX (and bearing inserted cysteines in HVR1 for position-specific PEGylation) were shown to evade antibody- and complement-mediated neutralization, as well as scavenger receptor-mediated uptake without loss of infectivity. Interestingly, despite a lack of the natural FX shield, PEGylation again improved transduction of hepatocytes as a function of PEG size36. However, it was shown that covalent shielding does have an impact on intracellular trafficking processes. Prill et al. compared irreversible versus bioresponsive shields based on pHPMA and demonstrated that neither the mode of shielding nor co-polymer charge had an impact on cell entry but did affect particle trafficking to the nucleus. Employing a bioresponsive shield with positively charged pHPMA co-polymers allowed for particle trafficking to the nucleus, maintaining the high transduction efficiencies of Ad vectors in vitro and in vivo37.
In summary, these data indicate that, even under the assumption that all vector-host-interactions were known and considered, excessive capsid surface modifications are necessary to overcome the hurdles associated with systemic vector delivery.
Here we provide a protocol to perform site-specific chemical modifications of adenovirus vector capsids for shielding and/or retargeting of adenovirus vector particles and adenovirus-based oncolytic viruses. The concept of this technology is outlined in Figure 1. It allows the shielding of certain capsid regions from unwanted interactions by covalent attachment of synthetic polymers. At the same, it also provides a means to attach ligands and combine shielding and targeting. Using simple chemistry, experimenters will be able to covalently modify adenovirus vector surface with a wide variety of molecules including peptides/proteins, carbohydrates, lipids, and other small molecules. Furthermore, the protocol provides a general concept for the chemical modification of biologically active virus-derived vectors under maintenance of their biological integrity and activity.

<table>
	<tbody>
		<tr>
			<td>Vector purification and chemical modification</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Argon gas</td>
			<td>Air liquide</td>
			<td>local gas dealer</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid Nitrogen</td>
			<td>Air liquide</td>
			<td>local gas dealer</td>
			<td></td>
		</tr>
		<tr>
			<td>500 mL centrifuge tubes</td>
			<td>Corning</td>
			<td>431123</td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup Express Plus 0.22 &#181;m</td>
			<td>Millipore</td>
			<td>SCGPU02RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(2-carboxyethyl) phosphine (TCEP)</td>
			<td>Sigma-Aldrich</td>
			<td>C4706-10g</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL (3mL) Norm Ject (syringes)</td>
			<td>Henke Sass Wolf</td>
			<td>4020.000V0</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine-Ject needles for single use (yellow 0.9 x 40 mm)</td>
			<td>Henke Sass Wolf</td>
			<td>4710009040</td>
			<td></td>
		</tr>
		<tr>
			<td>Caesium chloride 99.999% Ultra Quality</td>
			<td>Roth</td>
			<td>8627.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica gel beads</td>
			<td>Applichem</td>
			<td>A4569.2500</td>
			<td></td>
		</tr>
		<tr>
			<td>Methoxypolyethylene glycol maleimide - 750 (PEG mal-750)</td>
			<td>Iris Biotech</td>
			<td></td>
			<td>store in silica gel beads at -80 &#176;C</td>
		</tr>
		<tr>
			<td>13.2 mL Ultra Clear Ultracentrifuge Tubes</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td>only open in hood</td>
		</tr>
		<tr>
			<td>PD-10 size exclusion chromatography column</td>
			<td>GE Healthcare</td>
			<td>17-0851-01</td>
			<td>store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>AppliChem</td>
			<td>A1069.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS Ultrapure</td>
			<td>AppliChem</td>
			<td>A1112,0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>AppliChem</td>
			<td>A1123.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Material for cell-culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>PAN Biotech</td>
			<td>P04-36500</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>PAN Biotech</td>
			<td>P04-03590</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>PAN Biotech</td>
			<td>P10-0231SP</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS Good</td>
			<td>PAN Biotech</td>
			<td>P40-37500</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>PAN Biotech</td>
			<td>P06-07100</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosphere Filter Tips (various sizes)</td>
			<td>Sarstedt</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipettes (various sizes)</td>
			<td>Sarstedt</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>reaction tubes (various sizes)</td>
			<td>Sarstedt</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TC plates 15cm</td>
			<td>Sarstedt</td>
			<td>83.3903</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Material for silver staining protocol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>J.T.Baker</td>
			<td>8045</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>AppliChem</td>
			<td>1613,2500PE</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>AppliChem</td>
			<td>A0820,2500PE</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde 37%</td>
			<td>AppliChem</td>
			<td>A0877,0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>AppliChem</td>
			<td>A1613,2500PE</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium thiosulfate</td>
			<td>AppliChem</td>
			<td>1,418,791,210</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver nitrate</td>
			<td>AppliChem</td>
			<td>A3944.0025</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium carbonate</td>
			<td>AppliChem</td>
			<td>A3900,0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Special Lab Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Desiccator</td>
			<td>Nalgene</td>
			<td>5311-0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Megafuge 40</td>
			<td>Heraeus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Roter for Megafuge TX750 + Adapter andLlids for 500 mL tubes</td>
			<td>Heraeus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Conventional</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge e.g. Optima XPN-80</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>suitable Ultrazentrifuge Rotor e.g. SW41</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pH -Meter</td>
			<td>Conventional</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stand with clamps</td>
			<td>Conventional</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Goose neck lamp</td>
			<td>Conventional</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Over-head rotor</td>
			<td>Conventional</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal Block</td>
			<td>Conventional</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Photometer (OD 260)</td>
			<td>Conventional</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

combined genetic and chemical capsid modifications of adenovirus-based gene transfer vectors for shielding and targeting

Adenovirus vectors are potent tools for genetic vaccination and oncolytic virotherapy. However, they are prone to multiple undesired vector-host interactions, especially after in vivo delivery. It is a consensus that the limitations imposed by undesired vector-host interactions can only be overcome if defined modifications of the vector surface are performed. These modifications include shielding of the particles from unwanted interactions and targeting by the introduction of new ligands. The goal of the protocol presented here is to enable the reader to generate shielded and, if desired, retargeted human adenovirus gene transfer vectors or oncolytic viruses. The protocol will enable researchers to modify the surface of adenovirus vector capsids by specific chemical attachment of synthetic polymers, carbohydrates, lipids, or other biological or chemical moieties. It describes the cutting-edge technology of combined genetic and chemical capsid modifications, which have been shown to facilitate the understanding and overcoming of barriers for in vivo delivery of adenovirus vectors. A detailed and commented description of the crucial steps for performing specific chemical reactions with biologically active viruses or virus-derived vectors is provided. The technology described in the protocol is based on the genetic introduction of (naturally absent) cysteine residues into solvent-exposed loops of adenovirus-derived vectors. These cysteine residues provide a specific chemical reactivity that can, after production of the vectors to high titers, be exploited for highly specific and efficient covalent chemical coupling of molecules from a wide variety of substance classes to the vector particles. Importantly, this protocol can easily be adapted to perform a broad variety of different (non-thiol-based) chemical modifications of adenovirus vector capsids. Finally, it is likely that non-enveloped virus-based gene transfer vectors other than adenovirus can be modified from the basis of this protocol.

Figure 2 shows examples of the cytopathic effect (CPE) on 293 (HEK 293) cells that indicates successful vector production. Cells should show morphology (Figure 2C) 40-48 hours after inoculation with the virus vector. The right timepoint for harvesting is crucial for not losing virus particles by cell lysis and preventing oxidation of the genetically introduced thiol groups. If vector particles are released into the medium by cell...

Watch this Scientific Journal Video about Combined Genetic and Chemical Capsid Modifications of Adenovirus-Based Gene Transfer Vectors for Shielding and Targeting at JoVE.com

Combined Genetic and Chemical Capsid Modifications of Adenovirus-Based Gene Transfer Vectors for Shielding and Targeting

The protocol described here enables researchers to specifically modify adenovirus capsids at selected sites by simple chemistry. Shielded adenovirus vectors particles and retargeted gene transfer vectors can be generated, and vector host interactions can be studied.

Adenovirus vectors are potent tools for genetic vaccination and oncolytic virotherapy. However, they are prone to multiple undesired vector-host ...

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8.3K Views.  University Witten/Herdecke. The protocol described here enables researchers to specifically modify adenovirus capsids at selected sites by simple chemistry. Shielded adenovirus vectors particles and retargeted gene transfer vectors can be generated, and vector host interactions can be studied.

Video: Combined Genetic and Chemical Capsid Modifications of Adenovirus-Based Gene Transfer Vectors for Shielding and Targeting

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Annotation of Plant Gene Function via Combined Genomics, Metabolomics and Informatics

Given the ever expanding number of model plant species for which complete genome sequences are available and the abundance of bio-resources such as knockout mutants, wild accessions and advanced breeding populations, there is a rising burden for gene functional annotation. In this protocol, annotation of plant gene function using combined co-expression gene analysis, metabolomics and informatics is provided (Figure 1). This approach is based on the theory of using target genes of known function to allow the identification of non-annotated genes likely to be involved in a certain metabolic process, with the identification of target compounds via metabolomics. Strategies are put forward for applying this information on populations generated by both forward and reverse genetics approaches in spite of none of these are effortless. By corollary this approach can also be used as an approach to characterise unknown peaks representing new or specific secondary metabolites in the limited tissues, plant species or stress treatment, which is currently the important trial to understanding plant metabolism.

Combination of genomics, co-expression gene analysis and the identification of target compounds via metabolism give gene functional annotation.

Given the ever expanding number of model plant species for which complete genome sequences are available and the abundance of bio-resources such as ...

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

Combined Genetic and Chemical Capsid Modifications of Adenovirus-Based Gene Transfer Vectors for Shielding and Targeting

Summary

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Chapters in this video

Culture of myeloid dendritic cells from bone marrow precursors

Method for Culture of Early Chick Embryos ex vivo (New Culture)

Double Whole Mount in situ Hybridization of Early Chick Embryos

Method for Whole Mount Antibody Staining in Chick

Induction and Testing of Hypoxia in Cell Culture

Annotation of Plant Gene Function via Combined Genomics, Metabolomics and Informatics

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP