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).

플라스미드 pAdFTC 기반으로 재조합 HCAdV 게놈 1. 건설 참고 : 모든 플라스미드는 이전에 11, 12를 설명하고 요청에 따라 사용할 수있다. 클로닝 과정은도 1에 개략적으로 도시되어있다. <ol><li> pHM5-GOI를 생성하기 위해, 선택의 클로닝 전략을 사용하여, 셔틀 플라스미드 pHM5으로 프로모터 및 폴리아 데 닐화 신호 (PA)를 포함하여 관심 유전자 (GOI)를 복제. ...

<ol>
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여기에 제시된 프로토콜은 전술 한 절차에 기초 4,12 인간 아데노 바이러스 형 5에 기초 HCAdV 벡터들의 정화를 허용한다. pAdFTC 플라스미드 내의 HCAdV 게놈은 모든 아데노 바이러스 유전자의 결여 만 5&#39;- 및 3&#39;- LTR에 및 포장 신호를 전달한다. 이 전략에서 HV AdNG163R-2 (4)는 트랜스 효율적인 바이러스 생산에 필요한 모든 유전자를 제공한다. 또는 아데노 관련 바이러스 (AAV) 기...

유전자 치료 응용 것이 바이러스 단백질의 발현, 유전자 자체 또는 수신 바이러스 단백질에 의한 세포 독성 및 면역 부작용을 방지하기 위해 매우 중요하다. 아데노 바이러스 벡터 (ADV)가 널리 도입 유전자 발현 1,2의 영향을 조사하기 위해 다양한 세포에 외래 DNA를 도입하기 위해 사용된다. ADV의 가장 진보 된 버전은 모든 바이러스 코딩 서열 3,4-함으로써 낮은 면역 원성 및 저독성 5-8 결합 35킬로바이트까지 패키징을 제공하는 능력이 결여 된 고용량 아데노 바이러스 벡터 (HCAdV)으로 표시된다. 때문에 높은 포장 용량 그들은 하나의 벡터 용량을 사용하여 크거나 여러 유전자의 전달을 할 수 있습니다. 따라서, 그들은 연구 지역 사회를위한 유용한 도구를 나타냅니다. 대조적으로, 제일 - 또는 VEC를 2 세대 ADV 쉽게 상업용 키트를 사용하여 제조 할 수있다 초기 유전자 E1 및 / 또는 E3 결여 할토르 HCAdV의 게놈 구조와 바이러스 생산은 더 복잡하다. HCAdV 게놈의 건설을위한 시스템은 모든 바이러스 코딩 시퀀스 및 셔틀 플라스미드 pHM5 9-12없는 HCAdV 게놈을 운반하는 플라스미드 pAdFTC을 기반으로합니다. 최대 14 킬로베이스 (KB)의 관심의 모든 유전자가 여러 복제 사이트가 인식에 의해 형벌되는 셔틀 벡터 pHM5에 복제 할 수 있습니다 / 원점 복귀 엔도 뉴 클레아의 사이트를 절단 SceI 제한 I 및 I-CEU I.를 PI- 따라서, 관심의 유전자를 클로닝 연속 PI- 및 I-SceI 제한 I- CEU I 의해 플라스미드에 함유 pAdFTC HCAdV 게놈에 존재하는 동일한 제한 부위에 관한 후속 삽입 소화 해제 될 수있다. pAdFTC에 PI- SceI 제한 I 및 I- CEU 난 부위를 절단 사이에 유전자 삽입 부위는 스터 DNA 및 5 &#39;및 3&#39;반전으로 말단 반복 게놈 패키징에 필요한 비 암호화하는 아데노 바이러스 서열 (LTR에)와 측면으로 접하고단부와 하류 5&#39;ITR 패키징 신호 모두에서. 추가 투표자 DNA는 바이러스 생산시 효율적인 포장을 보장하기 위해 27-36킬로바이트까지 최종 HCAdV 게놈의 최적 크기를 제공합니다. pAdFTC이 (삽입 된 유전자의 크기에 따라 다름)까지 45킬로바이트와 큰 플라스미드와 비교적 긴 DNA 인식 사이트와 유도 엔도 뉴 클레아의 사용을하기 때문에 결합 강한 DNA, 몇 가지 정리 단계 pHM5에서 유전자의 전송시 필요한 전시 pAdFTC에. 전단력을 피하고 취급에주의하는 것이 좋습니다. HCAdV 게놈은 LTR에 5&#39;ITR의 상류에 직접적으로 위치되고 3&#39;ITR (12)의 하류 없음 I 제한 효소 인식 부위에 의해 측면된다. 따라서, HCAdV 없음 내가 HCAdV 생산 세포주에 바이러스 게놈의 후속 형질 다이제스트에 의해 방출 될 수있다. 참고 제한 효소의 사용하지 I REL위한플라스미드 pAdFTC에서 바이러스 게놈의 용이성 삽입 유전자가 없음 I DNA 인식 부위의 결여 된 것을 의미한다. HEK293 세포 기반 생산 세포 (116 세포)를 안정적으로 Cre 호텔 재조합 효소를 표현한다. 바이러스 증폭 셀 (116)은 트랜스 -3,4-의 복제 및 패키징을 위해 필요한 모든 ADV 유전자를 제공하는 헬퍼 바이러스 (HV)와 공동 감염. HV 116 4 세포에서 발현 Cre 호텔 재조합 바이러스에 의해 증폭되는 동안 제거 floxed 포장 신호와 제 세대 ADV이다. 이것은 본래 패키징 신호를 포함 주로 HCAdV 게놈 encapsidated되도록. HCAdV의 사전 증폭 조직 배양 접시의 표면에 성장 (116) 세포에서 시리얼 계대 단계를 수행하여 수행됩니다. 각각의 통과 후 바이러스 입자는 세 개의 연속 동결 - 해동 단계를 수행하여 감염된 세포에서 방출된다. 세포의 수가 증가는 모든 통로로 감염된선행 통로에서 세포 용 해물의 1/3. 마지막 사전 증폭 단계는 대규모 증폭 스피너 플라스크에 정지에서 재배 생산 세포를 감염하는 데 사용됩니다에서 마지막으로 해물. 비리 온은 4,12 구배 염화 세슘 밀도에서 초 원심 분리를 수행하여 현탁 세포로부터 정제된다. 이 절차를 빈 입자와 완벽하게 조립 입자는 두 가지 대역으로 분리된다. 상기 HCAdV을 집중하는 제 2 비 - 점진적으로 초 원심 분리 단계가 수행되는 입자를 포함한다. 이어서 HCAdV 함유 얻어진 밴드 수거하고 생리 완충액에 대해 투석한다. 최종 제제는 벡터의 절대 바이러스 입자 감염성 입자 및 HV 오염 수준의 숫자에 대한 특징이다. 절대 바이러스 입자는 바이러스 입자를 용균 및 260 nm에서의 흡광도를 측정함으로써 또는 정량적 실시간 PCR (qPCR에) (12)을 수행하여 결정될 수있다. 끝까지 마음의 감염ified하게 바이러스 입자는 감염된 세포에서 3 시간의 감염 후 내에 존재 qPCR의 측정 HCAdV 게놈에 의해 결정될 수있다.

<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>
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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.

복제, 증폭 및 HCAdV 준비 정화용 여기에 대표적인 예는 표시됩니다. 효소 소화 제한에 의해 복제 전략의 개요 (그림 1)과 복제에 대한 대표적인 예와 HCAdV 게놈의 릴리스는 (그림 2)이 제공된다. 소화 나는 PI- SceI 제한 I 및 I-CEU에 의해 pHM5에서 GOI-발현 카세트 및 후속 페놀 - 클로로포름 추출 및 EtOH로 침전의 출시 후 일반적인 제한 패턴 <...

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.

인간 아데노 바이러스 유형 5를 기반으로 대용량 아데노 바이러스 벡터의 클로닝 및 대규모 생산

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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