AAV (FLEX) Conditional Gene Expressi路匠on Vector (Cre-On)

The AAV (FLEX) conditional C鐵微re-On gene expression vector combin些章es VectorBuilder's要討 highly versatile AAV vector system wit報話h the Cre-responsive (FLEX) cond他信itional gene expression拿什 system to help you ach哥頻ieve AAV-mediated in vitro and in vivo個問 delivery of Cr有票e-responsive FLEX Cre-On switches. Th水站e FLEX Cre-On switch utilizes two你風 pairs of LoxP-variant reco電火mbination sites她動 flanking a gene of interest in時光 an arrangement which c他雜ompletely inhibits g市鐘ene expression in the ab劇視sence of Cre and ac樹店tivates gene expression upon Cre-de可明pendent inversi飛雜on of the coding sequence.

The FLEX Cre-On swit放西ch consists of two p秒刀airs of heterotypic票南 LoxP-variant recombin校門ation sites, namely LoxP, having請間 the wild type sequence and L內學ox2272, having a mutated sequence, 好人flanking an ORF which is in黃湖 the reverse (antisense) ori醫城entation relative to the promote文些r. Both LoxP va男購riants are reco議唱gnized by Cre, but章理 only identical pairs 行樂of LoxP sites can recombine with each睡煙 other and not with any other v姐頻ariant. The LoxP a鐘鐘nd Lox2272 sites are organized in an作樹 alternating fashion和銀, with an antiparallel orie火身ntation for each pair. In the absence o呢些f Cre recombinase, the討畫 ORF is not expressed due to its 件討antisense orientation relative to農日 the promoter. In the presence那離 of Cre, the LoxP and Lox2272 sites老什 undergo recombination with t到呢he other LoxP and Lox2272 s裡習ites respectively,訊呢 resulting in the inversi服上on of the ORF to a sense orientatio可什n and excision of o是火ne from each pair of iden來資tical recombination sites.但爸 This allows the u如理ser-selected promoter to drive我秒 the transcription of the gene of商要 interest. Since the ORF is now f唱服lanked by two different LoxP-variant 農但sites, no further recombination events 業一will take place even司店 when Cre is present.

An AAV vector is first constructed as都鐘 a plasmid in E. coli. It也地 is then transfected into暗是 packaging cells 爸南along with helper plasmi學姐ds, where the region of the 村兵vector between the two inverted日舞 terminal repeat見離s (ITRs) is packaged i道歌nto live virus. For the A農道AV (FLEX) conditiona了議l Cre-On gene expres拿紅sion vector, the FLEX Cre-On 多師switch described above is placed in-筆厭between the two ITRs dur兒分ing vector construction, 員河which is introduced in快現to target cells along舊女 with the rest of viral genome.兒信 Gene expression can then 議文be activated in t吧慢he presence of Cre recombi刀日nase upon Cre-mediated i也見nversion of the coding sequence.

The wild-type AAV genome is a linear s月藍ingle-stranded DNA (ssDNA) with two頻月 ITRs forming a hairpin structur讀關e on each end. It is therefore also 高著known as ssAAV. In懂間 order to express genes on ssAAV vecto場間rs in host cells, the ssDNA 間路genome needs to fi話友rst be converted to double-strande白看d DNA (dsDNA) through two pathways: 1)船如 synthesis of se唱長cond-strand DNA by the D件醫NA polymerase machinery of host對子 cells using the existing ssD也樹NA genome as the template and th文農e 3' ITR as the 東技priming site; 2) formation of inte車農rmolecular dsDNA between the plu來愛s- and minus-strand ssAA風少V genomes. The former pathw資女ay is the dominant one.

AAV genomic DNA forms episomal co答師ncatemers in the host cell nucleus.動爸 In non-dividing cells, these con笑媽catemers can remain fo影街r the life of the host cells. In divid妹現ing cells, AAV DNA is lost朋金 through the dilution effect of c音車ell division, beca吧唱use the episomal DNA doe是長s not replicate東好 alongside host cel分亮l DNA. Random inte如區gration of AAV DNA int票空o the host genom用男e can occur but is ex姐書tremely rare. T近能his is desirable in視吃 many gene therapy settings where t鐵數he potential oncogenic effe老從ct of vector integration 拍木can pose a significant concern.A major喝數 practical advantage of AAV is tha用他t in most cases A房音AV can be handled in biosafety lev醫坐el 1 (BSL1) fac計作ilities. This is due to AA但數V being inherently replication樹話-deficient, producing little 對明or no inflammation, and causing no 術們known human disease.空費 Due to their low immunogen刀爸icity in host org醫師anisms, AAV is the農媽 ideal viral ve城房ctor for many animal st什現udies.

Many strains of AAV have been iden快睡tified in nature機老. They are divided into different s影服erotypes based on diffe答兒rent antigenicity of the capsid protein購歌 on the viral surface. Different ser線算otypes can render the 務子virus with different tissue tropism (i路靜.e. tissue specificity朋哥 of infection). When our AAV 山地vectors are packaged 年鐵into virus, different serotypes can b媽答e conferred to the virus by using dif綠銀ferent capsid proteins for the packag在低ing. During cloning, I分農TRs from AAV2 are used,腦購 as this is common pr站空actice in the f雜報ield and does not impact specificity.弟這 Packaging helper plasmids incl關我ude a Rep/Cap plasmid, containing t我湖he replication genes from AAV2 and th站拍e capsid proteins for a chos遠西en serotype to determine tropism. Th草見e table below lists上煙 different AAV ser科喝otypes and their ti筆兵ssue tropism.

For further information about this vect是少or system, pleas妹熱e refer to the papers below個鐘.