Lentivirus Inducible 南好Gene Expression Vector (Tet-On)

VectorBuilder's lentivirus哥有 inducible gene expression鐵廠 vector combines the hi時森ghly efficient third-gen技輛eration lentiviral vector system w高電ith the Tet-On i得草nducible gene expressi月就on system to hel朋日p you achieve per妹的manent integration of tetracycline-in見師ducible gene express工商ion cassettes into the host genom金雨e.

The Tet-On inducible gene expression做呢 system is a powerfu上站l tool to control the timing of e一呢xpression of the gene(s) of interest (吧醫GOI) in mammalian cells. Our Tet-身美On inducible gene expressi就站on vectors are designed 是會to achieve nearly comple國在te silencing of a GOI in t火鐘he absence of tetr刀學acycline and its也關 analogs (e.g. doxycycline中廠), and strong, rapid expression in 都我response to the花外 addition of tetracycline or o理道ne of its analogs (e.g. doxycyclin車吧e). This is achieved through a m樹還ulticomponent system which inco問海rporates active silencing by the tTS 腦舞protein in the absence of高火 tetracycline an冷務d strong activation by但短 the rtTA protein in the為是 presence of tetracycline.&nb看歌sp;In the absence of tetracycline, 山劇the tTS protein derived f光笑rom the fusion of T件好etR (Tet repress朋亮or protein) and KRAB山道-AB (the transc裡路riptional repressor domain of Ki去著d-1 protein) binds to t草一he TRE promoter討件, leading t人計o the active suppression of gen議花e transcription. The rtTA protein, 短東;on the other hand你湖, derived from t現議he fusion of a mutant Tet repressor an做一d VP16 (the transcription 刀民activator domain of virion protein 16 視計of herpes simplex virus), binds to懂小 the TRE promoter to activate gen玩頻e transcription only in the山年 presence of tetracycline.

The lentiviral vec文照tor system is a highly ef司都ficient vehicle for intro林中ducing genes permanently into mammali友多an cells. A lentiviral vector i相從s first constructed as a plasm廠愛id in E. coli. For the 森煙lentivirus induci照愛ble gene expression vector, the tetr外秒acycline inducible e理區xpression cassette consisting 章文of the tetracycline inducibl雜低e element (TRE) promoter driving the 藍放GOI is placed in-between the two LT年廠Rs during vector construction. It is 年離then transfected into packaging cells a笑愛long with several helper plasmids. 匠腦Inside the packaging cells,制照 vector DNA located betw鐘校een the two long亮紙 terminal repeats (LTRs小吧) is transcribed into RNA, and問制 viral proteins expressed by 也銀the helper plasm很市ids further package慢城 the RNA into virus.些藍 Live virus is then relea低雜sed into the supernatant, w請答hich can be used to infect target cell也中s directly or after厭著 concentration.

When the virus is added to target c制公ells, the RNA cargo is shut為筆tled into cells where 線嗎it is reverse transcribe知的d into DNA and randomly integrate吃銀d into the host genome木音. The inducibl讀藍e expression cassette placed i計不n-between the two LTRs during vecto購在r construction are permanently 友做inserted into host DNA a爸章longside the rest of vir大說al genome. 

While our lentiv家物irus inducible gene expression vector少地 includes an inducible gen中舊e expression cassette consistin報放g of the TRE promoter driving the u慢站ser-selected GOI, the TRE binding regul鄉雜atory proteins rTS 視飛and rtTA have to be provided us南到ing a separate helper vector t有嗎o achieve tetra這費cycline induced gene exp資風ression in the presence of tetracycli農朋ne, while minimizing leaky expression 廠線in the absence of tetracycline. F鄉船or the lentivirus i有雨nducible gene expression vector syst土能em, the two-vector sys做東tem achieves higher le很兵vels of transgene induction i什笑n the presence of tetra文通cycline compared to an 農北all-in-one vector system. An all-in聽場-one vector con村站sists of two consecutive expre知文ssion cassettes: the GOI driven by T請也RE promoter and the tTS/rtTA genes dr南少iven by a ubiquitous or tissue-spe工兒cific promoter. 湖這For the lentiviral vectors, internal p雨討olyadenylation signal is not sugges睡月ted to be placed betw藍會een the LTRs for each individual expre日海ssion cassette, as this would inhibit 市房virus packaging. I對市nstead, a single polyade間廠nylation signal is placed in t區討he 3’LTR. As a result, transcriptio黃得n from the upstream TRE promoter of綠呢ten continues past the end通和 of the upstream ORF, through the dow弟謝nstream promoters and ORFs 新老;(tTS/rtTA genes). This often leads to 什女partial inhibition of expres吃子sion of the downstream t船議TS/rtTA, therefore preventing金唱 efficient induction of ge見紙ne expression in 視間the presence of tetracycline. Therefore自開, we recommend co-t朋知ransducing target ce腦音lls with lentiv中南irus carrying the TRE driven G那街OI and lentivirus expressing the t人討TS/rtTA cassette to achieve志男 the best induction ef請關ficiency.

By design, lentiviral刀美 vectors lack the genes required for vi空會ral packaging and transduction (the們民se genes are instead carried by雪筆 helper plasmids used during virus船如 packaging). As a resul拍道t, virus produced from l到靜entiviral vectors has the important sa厭鐵fety feature of being replicatio為喝n incompetent (meaning that th算謝ey can transduce target cells bu了懂t cannot replicate in the校厭m).

For further information abo秒說ut this vector syste兵音m, please refer to the papers below.男哥

Our Tet-On inducible gen長家e expression vectors are design服算ed to achieve nearly complete sile錯舊ncing of the GOI in the 民快absence of tetracy水嗎cline, and strong, rapid expression in 說低response to the addition 朋習of tetracycline. Th師作e lentiviral inducible gene expre服但ssion vector is derived from the thir家土d-generation lentiviral vector都這 system. It is op自白timized for high co朋妹py number replication in 票時E. coli, high-titer packaging of離黃 live virus, efficient viral t謝可ransduction of a醫鐘 wide range of cells, efficient vect短小or integration into the host geno線會me, and high-level transgene ex錯愛pression.

Limited cargo space: The wildtype些動 lentiviral genome is ~9.2報謝 kb. In our vector, the co如刀mponents necessary for viral pack業拍aging and transduc紙區tion occupy ~2.8 kb, which leaves ~6.4個國 kb to accommod計愛ate the user’s DNA of interest著南. When the vector goes beyond thi體外s size limit, viral titer can年拍 be severely reduced. The l女在entivirus inducible gene expression什國 vector is routinely 暗光used for inserting se遠會veral functional elements besides 問厭the ORF of the GOI, su劇去ch as the TRE promoter and dr討科ug resistance cassette. A l在哥arge ORF plus these addi明醫tional elements could ex和技ceed 6.4 kb, and the result could be 電城compromised viral producti文計on.

Technical complexity:&間件nbsp;The use of lentiviral vectors requ愛公ires the production of live v討放irus in packaging cells followed by數區 the measurement of viral titer動制. These procedures 多愛are technically demandin鐵女g and time consuming relative to con開道ventional plasmid tra雜書nsfection.