AAV (FLEX) Conditional Gene Expression 訊照Vector (Cre-Switch)

The AAV (FLEX) co弟討nditional Cre-Switch gene expression ve高討ctor combines VectorB藍年uilder’s highly versat西金ile AAV vector system with the Cre-resp公近onsive (FLEX) conditional gene 計也expression system to he不很lp you achieve AAV-mediated南嗎 in vitro and in vivo delivery 靜美of FLEX Cre-Switch for Cre-induced 章這switching between the expression of two開生 ORFs. The FLEX Cre個和-Switch system utiliz工問es two pairs of LoxP-variant reco鐘林mbination sites家雪 flanking two antiparallel商雨 ORFs in an arrangement which f的土acilitates acti黑費vation of one gene while repr藍少essing the other by Cre-dependent 討煙inversion of both ORFs.

The FLEX Cre-off switch c美如onsists of two pairs of快通 heterotypic LoxP-variant recombin暗但ation sites, namely LoxP大道 having the wild type sequenc船信e and Lox2272 having a muta費生ted sequence, flanki我錢ng an ORF. Both Lo笑個xP variants are recognized by Cre, but道輛 only identical pairs of LoxP 些市sites can recombine with ea呢雨ch other and not with any other varian藍資t. The LoxP and Lox2272 sites 書輛are organized in an來有 alternating fashion, with an anti那師parallel orientati醫的on for each pair. In the 遠裡absence of Cre recombina章大se, the ORF can be expr聽樹essed under the control of the上花 user-selected promoter. In the p信分resence of Cre, the LoxP and的請 Lox2272 sites undergo recombinati近樹on with the other 大你LoxP and Lox2272 sites respectively章金, resulting in the inve生黃rsion of the ORF to an an房還tisense orientation and excisio相雨n of one from each pair of identic影人al recombination si可司tes. Inversion of the ORF preven白草ts expression of the gene of interes頻區t. Since the ORF is no西可w flanked by two different LoxP-varian水男t sites, no further recombin文謝ation events will離愛 take place even when子答 Cre is present.

An AAV vector is fir麗不st constructed a北畫s a plasmid in E. coli. It is then光子 transfected into packaging ce中子lls along with helper plas從明mids, where the region of the vector be劇習tween the two i也月nverted terminal repeats (那玩ITRs) is packaged 唱錯into live virus. For the AAV (F嗎人LEX) conditional Cre-Switch gene expre老如ssion vector, th工山e FLEX Cre-Switch described above is 山拍placed in-between the two ITR你動s during vector construction, which is很農 introduced into target cell哥為s along with the rest of viral空生 genome. Expres爸關sion of the second ORF in th很章e FLEX Cre-Switch can then be ac答鐘tivated while silencing the first 月習ORF in the presence of Cre rec見頻ombinase, upon Cre-mediated in人也version of both O遠物RF sequences.

The wild-type AAV genome 家物is a linear single-stranded 關分DNA (ssDNA) with two ITRs form厭街ing a hairpin st窗志ructure on each end. It is therefore 快就also known as ssAAV. In ord知睡er to express genes南區 on ssAAV vectors in host cells, t身開he ssDNA genome needs 這子to first be converted to double-stran少用ded DNA (dsDNA) through two門一 pathways: 1) synthes路員is of second-strand DN信她A by the DNA polymerase machinery of 她低host cells using t自體he existing ssDNA genome as t公多he template and the 3' ITR as the primi大雨ng site; 2) formation of intermole但好cular dsDNA between the近體 plus- and minus-strand ssAAV 物畫genomes. The former pathway is the黃購 dominant one.

AAV genomic DNA forms episoma日大l concatemers in the host cell著媽 nucleus. In non-di低子viding cells, these co新綠ncatemers can remain for the li時水fe of the host cells. In dividing聽些 cells, AAV DNA is lost through the di內子lution effect of cell division, 科上because the episomal DNA does not rep大高licate alongside host cell DNA. 車老Random integratio快有n of AAV DNA into the host ge頻道nome can occur but is extremely業公 rare. This is desirable i紅下n many gene therapy settings where來麗 the potential oncogenic effect of 吧船vector integration ca呢請n pose a significant concern.公體A major practical advantage 弟歌of AAV is that in most case湖要s AAV can be handled in biosafety內船 level 1 (BSL1) facilitie小離s. This is due to AAV bein照窗g inherently replication-員業deficient, producing litt腦海le or no inflammation, and causing車子 no known human disease. Due to the有村ir low immunogenicity in host organi錢年sms, AAV is the ide木兒al viral vector for many animal stu章麗dies.

Many strains of AA區劇V have been identified in nature我跳. They are divided into如爸 different serotypes bas窗做ed on different antigenicity of the上鐵 capsid protein on 跳綠the viral surface. Different sero窗購types can render the virus with 可西different tissue tropism (i.e. tissu志能e specificity of infe會好ction). When our AAV vectors a暗市re packaged into virus,場花 different serotypes can be confer歌木red to the virus by using d快科ifferent capsid proteins for the pa和暗ckaging. During cloning, ITRs from A老在AV2 are used, as this is common prac說嗎tice in the field and樹靜 does not impact specif朋費icity. Packaging helper p飛相lasmids include a Rep/Cap plasmid, 站話containing the replic黑船ation genes from AAV2 and the c木器apsid proteins for a chosen s如明erotype to deter西睡mine tropism. The 電弟table below lists di自拍fferent AAV serotypes an少身d their tissue tropi師街sm.

For further informatio站坐n about this ve山家ctor system, please refer to t電都he papers below.