For details on the use of these cells with an adenox adenoviral system. Viruses were titered by the tcid 50 method on llcmk2 cells as described in the adenox expression system 1 user manual, pages 4647 august 2007 version, protocol pt34141, version pr7823350, clontech laboratories, inc. The recombination reaction was performed according to the instructions of the. System and provides instructions and guidelines to. Schematic diagram of the raav construct used in vitro and in vivo experiments is shown in figure a. However, in many situations, it requires the gene silencing in a. Rapad universal adenoviral expression system cell biolabs. Virapower adenoviral expression system takes advantage of the gateway recombination. The effects of gentamicin and penicillinstreptomycin on the electrophysiology of human induced pluripotent stem cellderived cardiomyocytes in manual patch clamp and multielectrode array system. The scrambled sequence of sigdnf, gt ctg ggt tca gtc act gta was designed and prepared in. The system uses a backbone vector from which the 5 itr, packaging signal and e1 sequences have been removed. In brief, a gene fragment was moved from th e pshuttle vector into the padenox. Invitrogens virapower adenoviral expression system facilitates highly efficient, in vitro or in vivo delivery of a target gene to dividing and nondividing mammalian cells using a replicationincompetent adenovirus.
The baculovirus system takes 56 weeks for production but has many advantages. Preparation of adenoviruses was carried out according to the adenox expression system 1 user manual clontech, mountain view, ca. Purpose of this manual this manual provides detailed information necessary to generate expression constructs of your gene of interest into the aavancedtm adeno associated virus aav cloning and expression vectors. Use the crisprcas9 smartnuclease system to edit the genome. Us20110201088a1 production of raav in vero cells using. Preparation of adenoviruses was initiated by transfection of recombinant adenoviral constructs into hek293 cells using lipofectamine 2000 according to the adenox expression system 1 user manual. Because the termini of the excised expression cassette fragment are compatible with the cloning site in adenox viral dna, the cassette can be directly ligated to adenox dna to form a recombinant adenoviral vector.
We describe a system based on a stably transformed insect cell lines harboring helper genes required for vector production. User manual adeno x rapid titer kit user manual united statescanada 800. Viruses were amplified in hek293 cells, purified using the adenox virus purification kit, and titered based on green fluorescent proteinvisualized. Widespread and targeted gene expression by systemic aav. Adenoassociated virus expression systems for gene transfer. Us7943379b2 production of raav in vero cells using. Full sequence for padenoxprlsdsredexpress linear shared on benchling. Therefore, users should assess transgene expression from a series of doses and at several. The htg edgeseq chemistry is compatible with fixed tissue samples, biofluids, and cell lines. An inducible system for highly efficient production of. Dilute adeno x system 1 viral dna or pshuttlelacz to 1 ngl.
Max adenovirus expression system for maximum adenovirus production. Aavrelatedexperimentsshouldbeconductedinbiosafetylevel2facilitiesbl2level. The htg edgeseq mirna whole transcriptome assay mirna wta enables users to measure the expression of 2,083 human mirna transcripts using nextgeneration sequencing ngs. Transfect the padcmvv5dest or padpldest expression construct into the 293a cell line to produce an adenoviral stock. Adeno x expression system 1 user manual pt3414 1 adeno x adenoviral system 3 user manual pt5177 1 adeno x 293 cell line protocolataglance cell type information the adeno x 293 cell line is a lowpassage, transformed human embryonic kidneyderived cell line selected for efficient adenovirus production and high transfection efficiency. Delivering plasmid vectors into cells by conventional transfection is technically straightforward, and far easier than virusbased vectors which require the packaging of live virus. The removal of these two viral genes creates space for.
Adenox expression system 1 knockout adenoviral rnai system 1 adenox adenoviral system 3 cmv virapower padcmvdest rapad cmv adenoviral expression systems mirna expression systems. The adenox rapid titer kit provides a complete set of materials for the quantification of adenoviral stocks. Advanced inducible gene expression systems user manual. The kit can be used to determine titers of the recombinant adenovirus created with our. Restriction map and multiple cloning site mcs of pshuttle vector.
Aavdj helper free promoterless expression system add to cart. Adenoassociated virusmediated gene transfer into rat carotid arteries. Adenoassociated virusmediated ilk gene silencing in the. Based on the secondgeneration vectors developed by bett et al. Aavdj helper free promoterless expression system, 1 kit. Construction of e1substituted adenoviral vectors using the adenoquick1. Viruses were titered by the tcid 5o method on llcmk2 cells as described in the adenox expression system 1 user manual, pages 4647 august 2007 version, protocol pt3414, version pr7823350, clontech laboratories, inc. Virapower adenoviral expression system thermo fisher. Adenoviral modulation of the tumorassociated system l.
Virapower adenoviral expression system thermo fisher scientific. I recently transduced hek293 cells 12well plate setup with the primary stock virus 0. Titration of adenoviral stocks is important for maintaining consistency between experimental samples and achieving the correct level of expression. Chromatography purification of canine adenoviral vectors. Guide to adenoviral components and vector construction. Regular plasmid gene expression vector vectorbuilder. Components of the virapower adenoviral expression system the virapower adenoviral expression system facilitates highly efficient, in vitro or in vivo delivery of a target gene to dividing and nondividing mammalian cells using a replicationincompetent adenovirus. Three aav serotypes aav2, aavdj and aav5 were precipitated using aavancedtm concentration reagent.
Constructing recombinant adenovirus with the adenox expression system 1. Cell biolabs rapad adenoviral expression system provides a much faster and safer method to generate rcafree recombinant adenovirus at high titer see table 1. Isolate 5080 ug of plasmid dna in less than 30 minutes. Adeno1 expression system enables efficient construction of recombinant adenoviruses.
Since the discovery that small interfering rna sirna can silence gene expression in mammalian cells elbashir et al. Htg edgeseq mirna whole transcriptome assay mirna seq. The virapower adenoviral expression system facilitates highly efficient, in vitro or in vivo delivery of a target gene to dividing and nondividing mammalian cells using a replicationincompetent adenovirus. Aav1 helper free bicistronic expression system gfp catalog number. Pdf chromatography purification of canine adenoviral vectors. Recombinant sequences were confirmed before subcloning into the adenoviral vector adenox clontech, mountain view, ca. Adenovirus was prepared according to the bd adenox expression system 1 user manual clonetech. User manual adenox rapid titer kit user manual united statescanada 800. The pcr assay was then repeated on the new viral stocks. The efficacy of commonly used drugs to treat adenovirus infections i.
Plasmid purification, isolation, extraction, plasmid miniprep, midiprep, maxiprep, endofree plasmid isoaltion, purification, fungal gdna, isolation, purification, blood rna isolation, blood genomic dna isolation, soil dna purification, plasmid megaprep, gigaprep, plasmid production service, 96well plasmid miniprep, m purifcation. Inordertobetterimprovetheinfectionefficiencyandspecificity. Isolation of highly infectious and pure adenoassociated. The expression cassette of interest is cloned into the multiple cloning site mcs of pe1. By eliminating the requirement for live helper virus the aav helperfree system provides a safer, purer and more convenient alternative to retroviral and adenoviral gene delivery systems. The plasmid backbone only occupies about 3 kb, leaving plenty of room to accommodate the users sequence of interest. The system is described in the following publication. Production of clinicalgrade gene therapy vectors for human trials remains a major hurdle in advancing cures for a number of otherwise incurable diseases. Purpose of this manual this manual provides detailed information necessary to generate expression constructs of your gene of interest into the aavancedtm adenoassociated virus aav cloning and expression vectors. Insect cells as a factory to produce adenoassociated virus type 2 vectors human gene therapy. This kit has been developed for use with any adenoviral system, provided that the hexon protein is being expressed. Adenox expression system 1 user manual pt34141 adenox adenoviral system 3 user manual pt51771. Pr5x1126 3 adenoxtm virus purification kit user manual i.
Pdf doxorubicin activates hepatitis b virus replication. The viral supernatants produced by transfecting 293 cells with recom binant padenox viral dna could. Because the termini of the excised expression cassette fragment are compatible with the cloning site in bd adenox viral dna, the cassette can be directly ligated to bd adenox dna. The adenoviral genome is established as an episome in the host cells nucleus, but is neither replicated nor. Max technology was developed by genetically modifying virus packaging cell, hek293 cell and adenoviral shuttle vector or adenoviral genome for maximum adenovirus production.