Adeno-Associated Virus 舊廠Tet Inducible Gene Ex黑體pression Vector

VectorBuilder’s adeno-associated 輛銀virus (AAV) Tet inducible 些廠gene expression vector老暗 combines the highl麗弟y versatile AAV vector system and the T土公et inducible gene expressi都弟on system to help you achieve AAV-med樹弟iated in vitro and in vivo delivery o開什f tetracycline-inducible gene express低做ion cassettes.

The Tet-On induc物做ible system is a powerful tool如微 to control the timing of exp南討ression of the gene(s) o紅老f interest (GOI) in mammalian cells.門房 Our Tet-On indu跳筆cible gene expression vec照店tors are designed to achie森東ve nearly complete silenc城資ing of a GOI in the absence of tetra船好cycline and its analogs (e.g算廠. doxycycline), and strong美國, rapid expression 草能in response to the addition of t畫跳etracycline or one 能也of its analogs (e.g. d農視oxycycline). Thi冷雜s is achieved through a multicomponen拍車t system which incorporates active sile了對ncing by the tTS p校你rotein in the absenc花林e of tetracycline and strong房熱 activation by the rtTA protein錢爸 in the presence東樹 of tetracycline. In the ab紅視sence of tetracycline, the tT他窗S protein derived from the fusio影呢n of TetR (Tet repressor protei公視n) and KRAB-AB (the transcripti靜東onal repressor 農車domain of Kid-1 protein) binds to the 紙國tetracycline-re你爸sponsive element (TRE) promoter, 科金leading to the active suppression姐資 of gene transcription. 自湖The rtTA protein, on the other ha熱很nd, derived from the fusion o去通f a mutant Tet repressor and VP16 (th請雜e transcription activator domain of vir麗城ion protein 16 of he得謝rpes simplex virus), binds t劇北o the TRE promot一飛er to activate gene transcrip那文tion only in the presenc短機e of tetracycline.

While our AAV Tet inducible gene expres年拍sion vector includes a光就n inducible gene expression cassette 習議consisting of the TRE pro志件moter driving t看家he user-selecte但著d GOI, the TRE binding regul黑你atory proteins tTS 資著and rtTA have to be pr低大ovided using a sepa村能rate helper vector t離老o achieve tetracycline induced票看 gene expression in the pres問信ence of tetracycline, while m筆草inimizing leaky expressio鄉船n in the absence of tetr銀笑acycline. 

The AAV Tet inducible gene事他 expression vector is 草費first constructed as a plasmid in E頻山. coli. It is then transfected into pac道紅kaging cells along with helper pl她街asmids, where the r年站egion of the vector betw新這een the two inverted termin金低al repeats (ITRs) is packaged into liv國明e virus. The tetracycline inducib飛音le gene expression cassette consisting海花 of the TRE promoter 朋影driving the user-selected GOI is外舊 placed in-between the two ITRs, wh就新ich is introduced 文聽into target cells along with the res習街t of viral genome. Gene expression作能 can then be turned on河作 in the presence of the rtTA regula爸話tory protein and tetracyc視校line.

AAV is effective看外 in transducing 一作many mammalian cell types,東媽 and, unlike adenovir笑頻us, has very low immunogenici綠的ty, being almost entire商樂ly nonpathogenic 嗎動in vivo. This makes the AAV inducible g分姐ene expression vector the idea子話l viral vector system for女人 achieving inducible gene e從頻xpression in vivo.

The wild-type AAV genome is a輛電 linear single-stra要對nded DNA (ssDNA) with two ITRs fo服西rming a hairpin structure on each通弟 end. It is therefo你草re also known as ssAAV. In orde司讀r to express genes on街女 ssAAV vectors i飛看n host cells, the ssDNA g銀他enome needs to 如議first be converted to double-stran外愛ded DNA (dsDNA) 兵說through two pathways: 1) sy笑票nthesis of second-str歌知and DNA by the DNA polymerase mac站什hinery of host cells using the ex動務isting ssDNA genome as the 飛雨template and the 3' ITR as the 技個priming site; 2)木喝 formation of intermo光資lecular dsDNA between the pl湖森us- and minus-str車作and ssAAV genomes. The former pathwa影拿y is the dominant one.

AAV genomic DNA fo銀森rms episomal concatemers in the host ce電如ll nucleus. In non-divi喝我ding cells, these concatemers c外站an remain for the life of th樂鐘e host cells. In d樂線ividing cells, AAV DNA is lost through 你離the dilution effect 錢熱of cell division, because the epi長姐somal DNA does not replicate al醫時ongside host cell DNA. Random integra河河tion of AAV DNA into the host genome街報 can occur but is ext化錯remely rare. The lack of integr新做ation is desirable in many gene therapy月還 settings where the potent學費ial oncogenic ef歌去fect of vector integration can 音中pose a significa從機nt concern.

A major practical ad風好vantage of AAV is that in most 空地cases AAV can be handled in biosaf嗎樹ety level 1 (BSL1) facilities. T船著his is due to AAV being inherently re的討plication-deficient, pr聽裡oducing little or no書匠 inflammation, and cau刀讀sing no known human disease.

Many strains of AAV hav車習e been identified in nature. T話工hey are divided into different se鐵文rotypes based on different 用答antigenicity of the capsid 商影protein on the viral s下音urface. Different serotype的自s can render the virus with differen媽市t tissue tropism (i.e. tissue spec好男ificity of infection). When our 是睡AAV vectors are packaged into virus, d但但ifferent serotyp玩城es can be conferred to the vi窗劇rus by using different capsid proteins小光 for the packaging. During clon山謝ing, ITRs from AAV2 are u光新sed, as this is co知票mmon practice in th頻兵e field and does not impact s年如pecificity. Packaging helper plasmid飛廠s include a Rep/Cap pl知車asmid, containing the replication gene綠鄉s from AAV2 and票音 the capsid protein很跳s for a chosen serotype to 美技determine tropism. Th西要e table below lists什吧 different AAV serotyp亮討es and their tissue tropism.

For further inform師刀ation about this vector議裡 system, please refer to the papers 劇北below.

Small cargo space: AAV has the smallest cargo capac報書ity of any of our viral vector systems物畫. AAV can accom民廠modate a maximum of 4.7 kb of sequenc黑城e between the ITR謝雜s, which leaves ~空風4.2 kb cargo space for user's DNA of i能多nterest.

Difficulty transducing c西和ertain cell types:男金 Our AAV vector system can t去爸ransduce many dif我火ferent cell types議地 including non-dividin照舞g cells when pack道可aged into the appropriate seroty習門pe. However, different AA友外V serotypes have tro關雨pism for different cell types明紙, and certain cell typ冷影es may be hard to t雨下ransduce by any serotyp資嗎e.

Technical complexity: The use of viral vectors require商月s the production of live virus in pack現睡aging cells followed by the measu機市rement of viral titer. These procedure制話s are technical現化ly demanding and time co身文nsuming relative to conventiona道木l plasmid transfection. These dem金風ands can be alleviated by ch光遠oosing our virus packaging services whe子還n ordering your vector.

5' ITR: 5' inverted terminal repeat. 兒鐘In wild type vi司自rus, 5' ITR and 3' ITR are市道 essentially ident又為ical in sequence. They reside on two影白 ends of the viral genom短北e pointing in opposite directions,得子 where they serve 不和as the origin of vira日拍l genome replication公見.

Promoter: The TRE promoter driving your gene of 村區interest is placed here.技事 

Kozak: Kozak consensus sequ慢可ence. It is placed in front o綠綠f the start codon of the ORF of金友 interest to facilitate trans謝工lation initiation些購 in eukaryotes.

ORF: The open reading frame好答 of your gene of interest is 鄉冷placed here.

Regulatory element: Allows the user討草 to add the Woodchuck hepatitis 樂話virus posttranscriptional r錯妹egulatory element (WPRE). WPRE e南美nhances virus stability 校河in packaging cells, leading to hi風音gher titer of packa南放ged virus; enhances higher expression o購也f transgenes.

BGH pA: Bovine growth hormone po空要lyadenylation. It facilitates t愛場ranscriptional terminati制們on of the upstream OR花場F.

3' ITR: 3' inverted terminal repeat. See電裡 description for 5’ ITR.

Ampicillin: Ampicillin resistance gene. It allows男員 the plasmid to新國 be maintained by少河 ampicillin selection in E. coli.著美

pUC ori: pUC origin of replication. Plasm長司ids carrying this origin exist in hi光都gh copy numbers in E. coli.