Subsequent efforts aimed at reducing process and productrelated impurities have been reported fig. The following day, when the cells grew up to 90% confluency. The 293 line of human embryonic kidney cells encodes the e1 region of the ad5 genome2. After three days of incubation, crude supernatants were recovered from the cells by freeze thaw lysates and. Generation and characterization of chimeric recombinant aav. The method, referred to as hot, is helper virus free, optically controllable, and based on transfection of only two plasmids, i. A plasmid is a small, circular piece of dna that is different than the chromosomal dna, which is all the genetic material found in an organism s chromosomes. Genetic modification of the escherichia coli strain dh5 to. Plasmid are of great use in biotechnology, they serve as vectors to amplify or express genetic information in foreign hosts. They are most commonly found as small circular, doublestranded dna molecules in bacteria. Plasmids can be found in all three major domains, archea, bacteria and eukarya.
These plasmids will typically contain a multiple cloning site mcs to insert the desired sequence, an origin of replication ori to duplicate in the host and selection mechanisms e. These genes e4, e2a and va mediate aav replication. New agrobacterium helper plasmids for gene transfer to plants. We present a simple and safe strategy for producing hightiter adenoassociated virus aav vectors derived from six different aav serotypes aav1 to aav6. This is one of four transfection protocols you can find in this web page set. In comparison to conventional helper plasmids, we characterize our constructs for aav protein expression, as well. Virus vector production using introncontaining helper plasmids lei cao, yuhong liu, matthew j. New agrobacterium helper plasmids for gene transfer to plants elizabeth e. The aav helper free system produces recombinant aav containing your gene of interest without the need to use a helper adenovirus. This is due to their ability to transduce noncycling cell populations and to integrate their genome into the host cell chromosome, resulting in the stable genetic modification of the transduced cell. The aav helper free system can accommodate inserts of up to 3 kb. They can only be transferred with the help of conjugative plasmids. Barnes prk20 mobilization helper plasmid for rk2orcolelderived plasmids, km 12 phc79 cosmidvector, ap, tc 18.
Mobilization of closely related plasmids pub110 and pbc16 by. L b selinger, n f mcgregor, g g khachatourians, m f hynes journal of bacteriology jun 1990, 172 6 32903297. Ep97499b1 aav helper plasmids for helper virusfree. Similar to viruses, plasmids are not considered a form of life as it is currently defined. We describe the construction and characterization of retroviral vectors and packaging plasmids that produce helperfree retrovirus with titers of 1 x 106 to 5 x 106 within 48 h. Briefly, bsr cells was seeded into wells of a 6well tissue culture plate and cultured at 37c for 24h. The aav vector packaging plasmids for the pseudotyped aav particles comprise the following dna sequences. Pcrbased reverse genetics strategy for bluetongue virus recovery.
Choose one of these plasmids if you already have the aav helper free system and would like to try a different aav serotype. Helper virusfree, optically controllable, and twoplasmid. Ex vivo cell therapy process using lentivirus as the vector to deliver the therapeutic transgene. The binary vector is a shuttle vector, socalled because it is. Melchers 3 and andre hoekema 3 1departmentof biology, utah state university, logan. Us20040209364a1 aav vector packaging plasmid for producing. At 96 h posttransfection, the cells were harvested and the cell pellets were collected. Browse aldevrons product overviews, brochures, guides and posters to learn more about our biotechnology products and services. The full text of this article is available as a pdf 216k. Bacterial plasmids plasmids are defined as double stranded, extrachromosomal genetic elements that replicate independently of the host cell chromosome and are stably inherited. Replication competent helper functions for recombinant aav. Helper plasmids were cotransfected with vector plasmids and mini adenovirus plasmid pf. Helper plasmids for production of hiv1derived vectors. These aav helper plasmids comprise the following dna sequences.
A protocol for aav vector production and purification. Our aav repcap plasmids work with our aav helper free systems to produce recombinant aav more safely and easily, without the need for a helper virus. The removal of the aav rep and cap genes allows for insertion of a gene of interest in the viral genome. Plasmids capable of integration into the chromosome were earlier called episomes. These vectors contain the immediate early region of the human cytomegalovirus. The cells were harvested 4 days after transfection. Plasmids used in lentiviral vector production, illustrating the plasmids common to all lentiviral vectors.
Activity oftdna borders plant cell transformation by plasmids. From itrbearing cis plasmids to very large helper plasmids, our manufacturing technology produces high yields even for the most difficult constructs. Jul 06, 2004 vectors derived from human immunodeficiency virus type 1 hiv1 appear an attractive option for many gene therapy applications. The starting material provided was predominantly dimerizedmultimeric. Mobilization of closely related plasmids pub110 and pbc16 by bacillus plasmid pxo503 requires transacting open reading frame beta. To package an aav vector of serotype 2, 3, or 6 into capsids of any aav serotype, 293t cells are cotransfected with the vector plasmid and the pdf plasmid expressing the desired cap gene. Feb 06, 2011 plasmids can plasmids are considered transferable genetic elements, or replicons, capable of autonomous replication within a suitable host. Molecular design for recombinant adenoassociated virus raav. The vector plasmid, helper plasmids mix, and adenovirus helper plasmids were cotransfected into human 293 cells. Genetic modification of the escherichia coli strain dh5. Pdf transmission of ciprofloxacin resistance in salmonella. In the aav helper free system, the rep and cap genes have been removed from the viral vector that contains aav2 itrs and are supplied in trans on the plasmid paavrc. They are artificial vectors that have both been created from the naturally occurring ti plasmid found in agrobacterium tumefaciens.
A transfer dna t dna binary system is a pair of plasmids consisting of a binary plasmid and a helper plasmid. We describe the construction of new helper ti plasmids foragrobacteriummediated plant transformation. Complete expression system contains packaging plasmids, expression vector and gfp control vector to produce recombinant aav serotype 6. The tdna regions of these plasmids were deleted using sitedirected mutagenesis to yield replicons.
May 17, 2018 comparative nucleotide sequence analysis of pih3 revealed a high degree of similarity with plasmids wz85p2 43, p6200114. Alla karpovas lab contains the inserts rep and raav2retro cap and is published in neuron. Cell therapy with lentivirus standardized plasmids for lentivirus aldevron paldlenti technical data 1b 1a 2a. In particular, we present a set of six novel helper plasmids, which upon simple cotransfection with an aav vector construct allow helper virusfree and highly ef. These plasmids are derived from three differentagrobacterium tumefaciens ti plasmids, the octopine plasmid ptib6, the nopaline plasmid ptic58, and the l,lsuccinamopine plasmid ptibo542. Aav vector and helper plasmids were transfected in equimolar amounts, i. In addition to bacterial chromosome nucleoid, bacterial cells normally contain genetic elements in their cytoplasm. The use of viral vectors in research is beneficial for a number of reasons, including but not limited to.
Plasmids used in aav viral vector production illustrating the helper plasmid, common to all raav vectors. Aav 6 repcap plasmid use one of our aav repcap plasmids along with the aav helper free system to produce recombinant human adeno associated virus raav virions without the need for a helper virus. In addition to rep and cap, aav requires a helper plasmid containing genes from adenovirus. Plasmids are mainly found in bacteria, but they can also be found in archaea and multicellular organisms. Plasmids, cloning vectors, shrna, cdna, orf clones. When a helper plasmid that encodes the e2a, e4, and va regions ad helper plasmid is used to transfect 293 cells, together with plasmids that encode.
We describe the construction and characterization of retroviral vectors and packaging plasmids that produce helper free retrovirus with titers of 1 x 10 6 to 5 x 10 6 within 48 h. During, and weidong xiao cns gene therapy center, department of neurosurgery, thomas jefferson university, philadelphia, pennsylvania 19107 received 27 june 2000accepted 14 september 2000. Despite design improvements, per cell productivity was lower than wildtype aav viral yields clark2002. The two plasmids are used together thus binary to produce genetically modified plants. A plasmid is a small, extrachromosomal dna molecule within a cell that is physically separated from chromosomal dna and can replicate independently.
The adenoviral genes required for proper aav packaging are provided in the phelper plasmid e2a, e4 and va rna or in the 293 packaging cells e1. The minimum regions in helper adenovirus that mediate the replication of the aav vector are e1, e2a, e4, and va1. These vectors contain the immediate early region of the human cytomegalovirus enhancerpromoter fused to the moloney murine leukemia virus long terminal repeat at. New agrobacterium helper plasmids for gene transfer to. Plasmid definition, types and functions biology dictionary. A desktop resource 1st edition 3 p a g e table of contents page chapter 6 chapter 1. In vivo gene therapy process using adeno associated virus aav as the vector to deliver the therapeutic transgene. Nov 25, 2004 0001 the present invention relates to aav helper plasmids for the helper virusfree packaging and pseudotyping of aav vectors. Aav helper plasmids for helper virusfree packaging and. Hightiter, wildtype free recombinant adenoassociated virus.
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