Single-Stranded A見朋AV (ssAAV) Tet Regulatory近體 Protein Expression Vect鐵很or

VectorBuilder’s s算道ingle-stranded 朋煙adeno-associated vir計會us (ssAAV) Tet regulato西空ry protein expression vector can 快科be used for AAV-bas少熱ed delivery of Tet regulatory proteins 個妹(tTS, rtTA, etc.少喝) into mammalian林很 cells to help you achiev說文e tetracycline-inducible ex老窗pression of target genes placed 不時downstream of a tetracycline r日人esponsive-element (TRE) promoter.

The Tet-On inducible system is a po姐通werful tool to control the地照 timing of expression of the gen多鐘e(s) of interest (坐弟GOI) in mammalian cells. Our Tet-新道On inducible gen窗也e expression vectors are designed to a到請chieve nearly complete silencing of時票 a GOI in the absence of tetracycli就技ne and its anal近問ogs (e.g. doxycycline), and 北坐strong, rapid e白弟xpression in response to the addit錯店ion of tetracycline or one of its analo懂刀gs (e.g. doxycycline). T校問his is achieved through a mult短好icomponent system which incorporate大器s active silencing by the 農秒tTS protein in the absence o報跳f tetracycline and strong activatio劇我n by the rtTA protein in匠謝 the presence of tetracycline. In 理鐘the absence of tetracycline, the tTS很小 protein derive土自d from the fusion of T場制etR (Tet repressor protein) 朋學and KRAB-AB (the tr下高anscriptional repressor domain of Kid-線河1 protein) binds to the TRE promote遠們r, leading to the active su明做ppression of gene transcripti城聽on. The rtTA protein, on the o科笑ther hand, derived from the fusi快開on of a mutant Tet r還用epressor and VP16 (the trans風劇cription activator domain新民 of virion protein 16 of h爸慢erpes simplex viru近人s), binds to the TRE promo票拍ter to activate gene trans廠腦cription only in the presen什下ce of tetracycline.頻術

The ssAAV Tet regulatory protein 間數expression vector is first construct飛短ed as a plasmid in E. coli. 北你The Tet regulatory protein expre商日ssion cassette co森答nsisting of the自秒 Tet regulatory protein(s)女跳 driven a user-selected promoter is 訊行placed in between the two inverte微金d terminal repeats畫身 (ITRs). It is then transfected into 是師packaging cells along 火明with helper plasmid劇信s, where the Tet regulatory prot冷間ein expression cassette between the tw關著o inverted termi是輛nal repeats (ITRs) is package民市d into active virus. Any gene(s)很吧 placed in-betwe懂秒en the two ITRs ar又林e introduced into t森微arget cells along哥志 with the rest o飛話f viral genome.

While our ssAAV Tet regulatory prot少術ein expression vector includes an 弟看expression cassette consisting of the 商銀Tet regulatory protein(s) driven錯員 by a user-selected promoter,校子 the GOI driven by 看身the TRE promoter must短暗 be provided us美看ing a separate helper vecto謝日r to achieve tetracycline induced ge報自ne expression i慢動n the presence of t技北etracycline, while minim場用izing leaky expression in從銀 the absence of森舊 tetracycline.

AAV is effective in transducing man章章y mammalian cell types, and un區匠like adenovirus has very low 電日immunogenicity, being almost entire懂花ly nonpathogenic in v計現ivo. This makes 熱有the AAV inducible gene expressio少舞n vector the ideal viral 們志vector system for achieving inducib見玩le gene expression in vivo.

The wild-type AAV genome is a linear 低吧single-stranded DN吧如A (ssDNA) with two ITRs 術哥forming a hairpin struct老書ure on each end. It is therefore also k吧光nown as ssAAV. In order to express g銀近enes on ssAAV vectors in host c請鄉ells, the ssDNA genome needs to firs話船t be converted to double-strande問東d DNA (dsDNA) through two路資 pathways: 1) synthesis of second-海山strand DNA by the DNA polymerase 場慢machinery of host cells using the e紅黑xisting ssDNA genome as the template睡低 and the 3' ITR as the志秒 priming site; 2) forma去文tion of intermolecular dsDNA be舊從tween the plus- and暗短 minus-strand ssAAV genomes. T長學he former pathwa請黑y is the dominant one.

AAV genomic DNA forms episomal concate歌光mers in the host cell nucleus短場. In non-dividing ce中物lls, these conca山答temers can remain for the life of 又數the host cells. In dividing c匠熱ells, AAV DNA is lost t對來hrough the dilution effect of cell錯有 division, because the episom國林al DNA does not re地時plicate alongside host cell DNA. 討做Random integration of A少遠AV DNA into the host genome朋家 can occur but is extreme相習ly rare. The lack of inte裡妹gration is desirable 笑和in many gene therapy 拿民settings where the potential on雜刀cogenic effect of vector integration c訊低an pose a significant concern.

A major practical advantage of AAV is t在森hat in most case要電s AAV can be handled i雨答n biosafety level 1 (BSL1)湖我 facilities. Thi線和s is due to AAV being inherently repli劇身cation-deficient, produc術離ing little or no inflam麗費mation, and causing no known human d上懂isease.

Many strains of AAV 有上have been identified現東 in nature. They are divid藍作ed into different 站媽serotypes based on di錢事fferent antigenicity of the capsi子中d protein on the viral sur體刀face. Different se玩麗rotypes can render the virus with 慢雜different tissue tropism (i.e. 民了tissue specificity o光自f infection). When 的分our AAV vectors a拿鐘re packaged into vi國業rus, different serotypes can be confer木她red to the virus by using different站要 capsid proteins for the packaging. Dur讀高ing cloning, ITRs from AAV2 are use車亮d, as this is common雜對 practice in the field and does not錢技 impact specificity. Packaging hel哥遠per plasmids include a Re答算p/Cap plasmid, containing th雜路e replication genes from AAV近門2 and the capsi問慢d proteins for a chosen serotype 相關to determine tropism.不年 The table below lists different AAV s計見erotypes and their tissue tropism.山放

For further informat很這ion about this vector system, please re音快fer to the papers belo報們w.