AAV (FLEX) Conditional Gene Ex懂她pression Vector (Cre-Off)

The AAV (FLEX) conditional Cre-Off 睡她gene expression v大海ector combines Ve門資ctorBuilder’s highly versatile AA會在V vector system業船 with the Cre-resp爸近onsive (FLEX) condit醫能ional gene expression system to h些弟elp you achieve AAV-mediated in vi腦上tro and in vivo delivery of C城書re-responsive FLEX Cre-Off switches.多長 The FLEX Cre-Off玩章 switch utilizes two pairs o短銀f LoxP-variant recombination sites爸錯 flanking a gene of interest in an arr火車angement which fac些費ilitates robust inactiv兒劇ation of gene expres在朋sion by Cre-dependent inver雜醫sion of the coding sequen算區ce.

The FLEX Cre-Off switch cons了技ists of two pairs of heterotypic Lox店作P-variant recombination sites, namely 弟東LoxP having the wild type seq要門uence and Lox2272 having 也林a mutated sequence, flanking an ORF那船. Both LoxP variants are recogn體離ized by Cre, but onl友化y identical pairs of LoxP sites海科 can recombine with each othe些吃r and not with any美黃 other variant. The LoxP 能數and Lox2272 sites are房在 organized in an a文車lternating fashion, with an文呢 antiparallel orientation for ea靜她ch pair. In the absence of Cre recombi文靜nase, the ORF can be expres見音sed under the control of the user-算呢selected promoter. In the presence of C習用re, the LoxP and Lox2272事計 sites undergo recombination with the 拍看other LoxP and Lox2272 sites respec作間tively, resulting in離間 the inversion of the ORF to an anti拿制sense orientation and excision畫懂 of one from each pa廠友ir of identical recomb科船ination sites. Inversi看店on of the ORF prevents expres舊煙sion of the gene of interest. Since 通藍the ORF is now flanked by two different海刀 LoxP-variant sites, 裡風no further recombination events will電就 take place even when Cre is p黑家resent.

An AAV vector is first construc學遠ted as a plasmid i來看n E. coli. It is then transfected玩坐 into packaging 和海cells along with helper plasmi多還ds, where the region 什秒of the vector between th體站e two inverted terminal repeats (ITRs好業) is packaged into live 一知virus. For the AAV (FLEX月呢) conditional Cre-Off gene expre得文ssion vector, the FLEX 筆火Cre-Off switch described ab腦林ove is placed in-bet路水ween the two ITRs during vector constr間訊uction, which is introduced唱北 into target cell看文s along with the 市少rest of viral genome. Gene expression光去 can then be inactivated in the presen公你ce of Cre recombinase upon Cr市新e-mediated inversion of the 山懂coding sequence.

The wild-type AAV geno東照me is a linear single-stranded DNA道不 (ssDNA) with two 好喝ITRs forming a hairpin structure on ea站報ch end. It is therefore also known a些在s ssAAV. In order to 你下express genes on ssAAV vector妹弟s in host cells, the ssDN相外A genome needs to first be c畫遠onverted to double-stranded DNA些也 (dsDNA) through two path通他ways: 1) synthe金用sis of second-st店師rand DNA by the DN下舊A polymerase machi短書nery of host cells using the existing s但土sDNA genome as the template遠花 and the 3' ITR as the兒水 priming site; 2) formation of店街 intermolecular dsDNA between the 習謝plus- and minus-strand ssAAV genomes腦亮. The former pathway is the d那高ominant one.

AAV genomic DNA forms episomal conca媽不temers in the host c快懂ell nucleus. In non-divid他務ing cells, these conca師窗temers can remain for the lif風費e of the host cells雨錢. In dividing cell拍報s, AAV DNA is lost throu個農gh the dilution effec舊謝t of cell division, be河在cause the episomal DNA does 是路not replicate alongsid中河e host cell DNA. Random inte東用gration of AAV DNA into th熱子e host genome can occur 離鐵but is extremely rare. This is desir街身able in many gen睡年e therapy settings 師是where the potential oncogenic ef南國fect of vector integration 報兵can pose a significant concern.醫理

A major practical a河子dvantage of AAV is that 玩購in most cases AA開務V can be handled in bi間制osafety level 1 (BSL1) facilities. Thi爸鄉s is due to AAV bei上白ng inherently replication-deficient現人, producing little or no infla少報mmation, and causing快路 no known human disease. Due to their l鐘地ow immunogenicity 個通in host organisms, AAV is 西讀the ideal viral vecto腦上r for many animal studies.

Many strains of AAV have been identifie會西d in nature. They are divided into d會他ifferent serotypes based on diffe拿路rent antigenicity of th老白e capsid protein on the viral surfac錯訊e. Different serotypes can r事志ender the virus wit件下h different tissu藍事e tropism (i.e. tis道還sue specificity of infectio國師n). When our AAV vectors are packaged大藍 into virus, differ西花ent serotypes can be con美聽ferred to the virus by using differen弟知t capsid proteins for the火鐵 packaging. During 村師cloning, ITRs from AAV技南2 are used, as this 她紅is common practice in th道雨e field and does not impact specific技風ity. Packaging helper plasmi西河ds include a Rep/Ca頻計p plasmid, containing th見計e replication genes from A雪房AV2 and the capsid proteins for a鐵哥 chosen serotype to間大 determine tropi坐北sm. During cloning, ITRs現在 from AAV2 are used, as this is comm見來on practice in the field and does no費術t impact specificity. Packagin民都g helper plasmids include a術靜 Rep/Cap plasmid, cont舞問aining the replication genes f工問rom AAV2 and the capsid proteins都們 for a chosen serotype to deter能子mine tropism. The table below lists di現現fferent AAV serotyp分家es and their tissue tropism.

For further information地事 about this vector system, please綠花 refer to the papers below.

The AAV (FLEX) condit黑哥ional Cre-Off gene expression v頻下ector is designed to achieve Cre-medi雜著ated conditional inhibition廠物 of gene expression in mammalian c劇會ells and animals. E通視xpression of th鐵銀e gene of interest i雨算s initially under the control of t男舞he user-selected promoter and can秒劇 be permanently si呢習lenced by co-expression of Cr多亮e recombinase, which will inve錯議rt the gene of interest to its ant明吃isense orientation.

This vector is 近懂optimized for high cop請對y number replication in E.身刀 coli, high-titer packaging of live v報章irus, efficient舊呢 transduction of host cells, and hig唱報h-level transgene e理請xpression. This AAV vira內坐l vector can be pac高醫kaged into virus using all k時習nown capsid serotypes工謝, is capable of very high雜見 transduction efficiency, and presents信費 low safety risk.

Small cargo space: AAV has the smallest cargo capacity of 如上any of our viral vect數山or systems. AAV c低場an accommodate a maximum of 4.作區7 kb of sequence between the IT訊兵Rs, which leaves ~4.1 kb cargo space 志鄉for the user's DNA of interest in th看愛e AAV (FLEX) conditional Cre-Off gene e女鄉xpression vector.

Difficulty transducing路市 certain cell types用身: Our AAV vector system can transduce man拍坐y different cell types湖吧 including non-dividing c街村ells when packag輛計ed into the appropriate serotype. Howe道妹ver, different AAV serotypes have tr短能opism for different cell t外船ypes, and certain cell types may b拿爸e hard to transduce by any serotype.

Technical complexit間制y: The use of viral vectors requires黃短 the production of li件朋ve virus in packagin厭技g cells followed by這服 the measurement of 話車viral titer. These proced關為ures are technically demanding a關畫nd time consuming relative t話靜o conventional pl熱校asmid transfection. These demands can 子樹be alleviated by choosing ou算生r virus packaging services when ord校視ering your vector.