Name: efficient recombinant parvovirus production with the help of adenovirus-derived systems
Uploaded: 2012-04-23T12:00:00
Description: Watch this Scientific Journal Video about Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems at JoVE.com

우리는 특히 마커스 뮐러, 실비아 Münstermann, 바바라 Liebetrau와 맨디 Roscher에서 DKFZ 바이러스 생산과 개발 단위의 팀을 감사드립니다. 이 연구는 부분적으로 교육과 연구 (BMBF)와 Deutsches Krebsforschungszentrum / Cancéropôle 뒤 그랜드 - 예상 응용 Tumour 바이러스학의 공동 프로그램의 프레임 워크의 Helmholtz 협회의 연방 교육부에서 교부금에 의해 지원되었습니다.

biosafety 레벨 2 연구실은 재조합 parvoviruses (recPV)의 생산을 위해 필요합니다. 프로토콜은 두 가지 부분으로 세분화됩니다. 첫 번째 부분은 (transfection 통해 recPV 생산) 프로토콜의 두 번째 부분 (감염을 통해 recPVs 생산)에 inoculum 역할 recPVs의 최소한의 금액을 생산하는 데 필요합니다. 일단 recPVs의 작은 금액은 프로토콜의 첫 번째 부분을 생략할 수 있고, 생산하고 있으며, 재조합 ...

<ol>
	<li>Cotmore, S. F., Tattersall, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parvoviral+host+range+and+cell+entry+mechanisms.">Parvoviral host range and cell entry mechanisms.</a> Adv. Virus. Res. 70, 183 (2007).</li><li>Rommelaere, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncolytic+parvoviruses+as+cancer+therapeutics.">Oncolytic parvoviruses as cancer therapeutics.</a> Cytokine Growth Factor Rev. 21, 185 (2010).</li><li>Hristov, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Through+Its+Nonstructural+Protein+NS1,+Parvovirus+H-1+Induces+Apoptosis+via+Accumulation+of+Reactive+Oxygen+Species.">Through Its Nonstructural Protein NS1, Parvovirus H-1 Induces Apoptosis via Accumulation of Reactive Oxygen Species.</a> J. Virol. 84, 5909-5909 (2010).</li><li>Cornelis, J. J., Salome, N., Dinsart, C., Rommelaere, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vectors+based+on+autonomous+parvoviruses:+novel+tools+to+treat+cancer.">Vectors based on autonomous parvoviruses: novel tools to treat cancer.</a> J. Virol. 6, 193-193 (2004).</li><li>El-Andaloussi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+adenovirus-based+helper+system+to+support+production+of+recombinant+parvovirus.">Novel adenovirus-based helper system to support production of recombinant parvovirus.</a> Cancer Gene Ther. 18, (2011).</li><li>Kestler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cis+requirements+for+the+efficient+production+of+recombinant+DNA+vectors+based+on+autonomous+parvoviruses.">cis requirements for the efficient production of recombinant DNA vectors based on autonomous parvoviruses.</a> Hum. Gene. Ther. 10, 1619-1619 (1999).</li><li>Xiao, X., Li, J., Samulski, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+high-titer+recombinant+adeno-associated+virus+vectors+in+the+absence+of+helper+adenovirus.">Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus.</a> J. Virol. 72, 2224 (1998).</li><li>He, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+system+for+generating+recombinant+adenoviruses.">A simplified system for generating recombinant adenoviruses.</a> Proc. Natl. Acad. Sci. U.S.A. 95, 2509 (1998).</li><li>Zolotukhin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+adeno-associated+virus+purification+using+novel+methods+improves+infectious+titer+and+yield.">Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield.</a> Gene. Ther. 6, 973 (1999).</li><li>Maxwell, F., Harrison, G. S., Maxwell, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+production+of+recombinant+AAV+by+transient+transfection+of+NB324K+cells+using+electroporation.">Improved production of recombinant AAV by transient transfection of NB324K cells using electroporation.</a> J. Virol. Methods. 63, 129 (1997).</li><li>Wrzesinski, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+and+pseudotyped+parvoviruses+minimize+the+contamination+of+recombinant+stocks+with+replication-competent+viruses+and+identify+a+DNA+sequence+that+restricts+parvovirus+H-1+in+mouse+cells.">Chimeric and pseudotyped parvoviruses minimize the contamination of recombinant stocks with replication-competent viruses and identify a DNA sequence that restricts parvovirus H-1 in mouse cells.</a> J. Virol. 77, 3851 (2003).</li></ol>

우리는 recPV 생산은 adenoviral 게놈 요소의 존재에 의해 향상될 수있는 것으로 나타났습니다. 우리는 transfection 통해 아데노 바이러스 게놈 요소를 제공함으로써 관련 recPV와 함께 광고 - 부사장 - 도우미와 100 개 이상의 접이식 공동 감염 세포에 의해 10 개 이상의 배 (0.3-5 TU / 셀)에 의해 recPV 수율을 증가 기존의 프로토콜에 비해. 프로토콜은 더욱 아데노 바이러스 게놈 요소의 전달 및 감염에 사용하?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> DMEM </td><td> 시그마 </td><td> D5796 </td></tr><tr><td> MEM </td><td> 시그마 </td><td> M4655 </td></tr><tr><td> Foetal 소 혈청 </td><td> PAA </td><td> A15-101 </td></tr><tr><td> L-글루타민 </td><td> Gibco </td><td> 25030-024 </td></tr><tr><td> Fugene </td><td> 로슈 </td><td> 047097050001 </td></tr><tr><td> 트립신-EDTA (에틸렌 다이아 민 테트라 초산) </td><td> Invitrogen </td><td> 25300062 </td></tr><tr><td> Benzonase Nuclease </td><td> 시그마 </td><td> E8263 </td></tr><tr><td> Adeno-X 신속한 Titer 키트 </td><td> Clontech </td><td> 632,250 </td></tr></tbody></table>

efficient recombinant parvovirus production with the help of adenovirus-derived systems

같은 쥐 H-1PV 및 MVM 등 설치류의 parvoviruses은 (PV), 작은 icosahedral, 단일 좌초된, DNA 바이러스입니다. 그들의 게놈은 각각 한 비 구조 (NS1과 NS2)과 capsid 단백질 (VP1과 VP2)의 표현을 조절이 발기인 P4와 P38을 포함합니다. 그들은 oncolytic 및 oncosuppressive 능력에 대한 anticancer 대리인의 인간 2 비 병원성하면서로서 높은 관심을 끌. NS1 바이러스는 세포 독성의 3 대 이펙터입니다. 추가로 천연 antineoplastic 활동을 강화하기 위해서는 이러한 벡터로부터 파생은 치료 transgene (예 : 세포 독성 폴리펩티드, 시토킨, 케모카인, 종양 억제 유전자 등) 4 capsid 단백질에 대한 유전자 인코딩 교체에 의해 생성되었습니다. 재조합 parvoviruses (recPVs)는 벡터가 NS1 / 2 코딩 시퀀스 및 바이러스성 DNA의 증폭 및 패키징에 필요한 PV의 게놈 telomeres을 유지합니다. 생산recPVs 단지별로 생산 세포 (일반적으로 HEK293T)에서 발생하는 공동 transfecting 부사장 단백질에 대한 유전자 인코딩 (그림 1) 4을 표현 두 번째 벡터 (pCMV-VP)으로 세포를. 이렇게 생성된 recPV 벡터는 복제에 결함이 있습니다. recPVs 그들이 생성되어있는 부모의 바이러스에 대하여 강화된 oncotoxic 활동을 소유 입증했지만, 그들의 생산을 주요 과제 남아 강력하게 안티 - 암 임상 응용에 이러한 에이전트의 사용을 hampers. 우리가 사용하는 다른 프로토콜에 비해 이상 10 배 이상으로 recPVs의 생산을 향상 HEK293T로 E2a, E4 (orf6) 및 VA RNA의 유전자 (예 : pXX6 플라스미드)를 포함하는 광고-5 파생된 벡터의 도입을 발견. 이 연구 결과를 바탕으로, 우리는 recPVs 생산을 강화하는 데 필요한 게놈 adenoviral 요소뿐만 아니라 parvovir 포함되어 AD-VP-헬퍼 소설을 만들었저희 부사장 유전자 단위 5. AD-VP-헬퍼의 사용은, 레크 리에 - 베트남 후 증후군의 생산 ((예 NB324K) 가능 transfect하기 어려운 세포 라인의 활용, 바이러스성 감염 단계 (같은 플라스미드 transfection 반대)에 전적으로 의존 프로토콜을 사용하여 수 그림. 2). 우리는 크게 최종 제품 5 품질에 영향을 미치지 않고, 생산 시간 및 비용을 모두 절감, 생산 재조합 바이러스의 양을 향상 방법을 제시한다. 또한, recPV의 대규모 생산은 (현탁액 세포와 bioreactors에서) 지금 생각할 수있다.

Watch this Scientific Journal Video about Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems at JoVE.com

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

여기에는 오직 사용중인 다른 프로토콜에 비해 100 개 이상의 배 이상에 의한 재조합 파르 보 바이러스 생산의 효율성을 향상시켜 세포 감염에 기반 프로토콜을 설명합니다. 이 프로토콜은 파르 보 바이러스의 부사장의 전송 단위 (광고-VP)를 포함하는 5-기반 도우미 소설 아데노 바이러스의 사용에 의존하고 있습니다.

아데노 바이러스 파생 시스템의 도움으로 효율적인 재조합 파르 보 바이러스 생산

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 ...

efficient-recombinant-parvovirus-production-with-help-adenovirus

Research

JoVE Journal

Immunology and Infection

Estimating Virus Production Rates in Aquatic Systems

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative estimates of this process is critical as we can then develop better models of microbial community structure and function as well as advance our understanding of how viruses work to alter aquatic biogeochemical cycles. The virus reduction technique allows researchers to estimate the rate at which virus particles are released from the endemic microbial community. In brief, the abundance of free (extracellular) viruses is reduced in a sample while the microbial community is maintained at near ambient concentration. The microbial community is then incubated in the absence of free viruses and the rate at which viruses reoccur in the sample (through the lysis of already infected members of the community) can be quantified by epifluorescence microscopy or, in the case of specific viruses, quantitative PCR. These rates can then be used to estimate the rate of microbial mortality due to virus-mediated cell lysis.

The turnover rate of viruses in marine and freshwater systems can be estimated by a reduction and reoccurrence technique. The data allow researchers to infer rates of virus-mediated microbial mortality in aquatic systems.

아쿼틱 시스템에서 바이러스 생산 요금 예측

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative ...

연구

면역학 및 감염병학

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

B 세포의 재조합 생산 및 Retroviral 감염

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

세포 성숙없이 돌기 세포의 효율적인 Transfection을 위해 최적화된 프로토콜

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

Production and Titering of Recombinant Adeno-associated Viral Vectors

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced.

재조합 Adeno - 관련 바이러스성 벡터의 생산 및 Titering

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

파지 - 표시 합성 항체 라이브러리의 확장 성 높은 처리량 선택

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses targeting myelin. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model of CNS autoimmune disease, and is typically induced via immunization with short peptides representing immunodominant CD4+ T cell epitopes of myelin proteins. However, B cells recognize unprocessed protein directly, and immunization with short peptide does not activate B cells that recognize the native protein. As recent clinical trials of B cell-depleting therapies in MS have suggested a role for B cells in driving disease in humans, there is an urgent need for animal models that incorporate B cell-recognition of autoantigen. To this end, we have generated a new fusion protein containing the extracellular domain of the mouse version of myelin oligodendrocyte glycoprotein (MOG) as well as N-terminal fusions of a His-tag for purification purposes and the thioredoxin protein to improve solubility (MOGtag). A tobacco etch virus (TEV) protease cleavage site was incorporated to allow the removal of all tag sequences, leaving only the pure MOG1-125 extracellular domain. Here, we describe a simple protocol using only standard laboratory equipment to produce large quantities of pure MOGtag or MOG1-125. This protocol consistently generates over 200 mg of MOGtag protein. Immunization with either MOGtag or MOG1-125 generates an autoimmune response that includes pathogenic B cells that recognize the native mouse MOG.

We describe a simple protocol using only basic lab equipment to generate and purify large quantities of a fusion protein that contains mouse Myelin Oligodendrocyte Glycoprotein. This protein can be used to induce experimental autoimmune encephalomyelitis driven by both T and B cells.

실험자가 면역 뇌척수염 연구 간단하고 효율적인 생산 및 마우스 미엘린 희소 돌기 아교 세포 당 단백질의 정제

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

를 사용하여 림프구 혈관 외로 유출 분석<em&gt; 체외</em&gt; 인간의 혈액 - 뇌 장벽의 모델

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared to IN from other retroviruses, PFV IN is more soluble and more amenable to experimental manipulation. Additionally, it is sensitive to clinically relevant human immunodeficiency virus (HIV-1) IN inhibitors, suggesting that the catalytic mechanism of PFV IN is similar to that of HIV-1 IN. IN catalyzes the covalent joining of viral complementary DNA (cDNA) to target DNA in a process called strand transfer. This strand transfer reaction introduces nicks to the target DNA. Analysis of integration reaction products can be confounded by the presence of nucleases that similarly nick DNA. A bacterial nuclease has been shown to co-purify with recombinant PFV IN expressed in Escherichia coli (E. coli). Here we describe a method to isolate PFV IN from the contaminating nuclease by heparin affinity chromatography. Fractions are easily screened for nuclease contamination with a supercoiled plasmid and agarose gel electrophoresis. PFV IN and the contaminating nuclease display alternative affinities for heparin sepharose allowing a nuclease-free preparation of recombinant PFV IN suitable for bulk biochemical or single molecule analysis of integration.

Recombinant prototype foamy virus integrase protein is often contaminated with a bacterial nuclease during purification. This method identifies nuclease contamination and removes it from the final preparation of the enzyme.

검색 및 재조합 프로토 타입 거품 바이러스 Integrase의 정화 동안 Nuclease 오염의 제거

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared ...

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

CRISPR/Cas9와 재조합 조류 Herpesvirus 벡터 생성 편집 진

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian ...

Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 per codon, the actual error rates vary widely, depending on the species, environment, and codons being studied. We have previously shown that mycobacteria use a two-step pathway for the generation of aminoacylated glutamine and asparagine tRNAs and that this is specifically associated with relatively high error rates due to the modulation of mistranslation rates by an essential component of the pathway, the amidotransferase GatCAB. We modified a previously employed Renilla-Firefly dual-luciferase system that had been used to measure mistranslation rates in Escherichia coli for use in mycobacteria to measure specific mistranslation rates of glutamate at glutamine codons and aspartate for asparagine codons. Although this reporter system was suitable for the accurate estimation of specific error rates, lack of sensitivity and requirements for excessive manipulation steps made it unsuitable for high-throughput applications. Therefore, we developed a second gain-of-function reporter system, using Nluc luciferase and green fluorescent protein (GFP), which is more amenable to medium/high-throughput settings. We used this system to identify kasugamycin as a small molecule that can decrease mycobacterial mistranslation. Although the reporters that we describe here have been used to measure specific types of mycobacterial mistranslation, they may be modified to measure other types of mistranslation in a number of model systems.

In this article, we present two complementary methods to measure specific rates of translational error and mistranslation in the model mycobacterium, Mycobacterium smegmatis, using gain-of-function reporter systems. The methods can be employed to measure accurate error rates in low throughput or relative error rates in a more high-throughput setting.

기능 리포터 시스템으로 특정 진 균의 오 번역 비율 측정

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 ...