Lentivirus IPTG-Inducible s但路hRNA Knockdown Vecto北朋r

Our Lentivirus IPTG-Inducibl上女e shRNA Knockdown 小熱vectors are deri水物ved from the third-ge唱問neration lentivira這姐l vector system. This system is optimi些說zed for high copy number repli飛服cation in E. coli, hig近去h-titer packaging of live viru內工s, efficient viral trans又放duction of a wide range of cells, and e我山fficient vector integ長船ration into the ho音機st genome. The modified human U6 p身關romoter with two LacO ele筆吧ments drives high-level tran路作scription of the downstream shRNA in ma對科mmalian cells in the presence of I吧頻PTG, which preven小和ts LacI from bi路紅nding to LacO. Our shRNA stem-技習loop sequences are optimi微黃zed to mediate efficient shRNA proce道爸ssing and target gene knockdown.

Our lentivirus I就放PTG-inducible shRNA knock鐘刀down vector has been validated 錢章for highly efficient target gene knock購森down in the presence 小下of IPTG as shown in Figure 1 below.

Figure 1. EGFP knockdown with遠裡 the lentivirus IPTG-inducible shRNA ve厭黃ctor system. (A) L坐話entiviral vectors carr間街ying IPTG-induc我坐ible U6-based scramble or EG議雪FP-targeting sh月她RNA expression c快呢assettes were packaged into the cor店通responding lentiviral particles and tr線紙ansduced into HEK293T cel但光ls stably expressi慢喝ng EGFP. Antibiotic selecti見件on with appropriate antibiotics, 南到puromycin (Puro) or blasticidin (Bsd), 也森was performed to isol好要ate positively transd金遠uced cells follow廠志ed by treatment with 1mM IPTG音腦 to induce shRNA 訊跳expression. Median fluore樂哥scence intensity (MFI) of EGFP 都購was quantified for all experimental g到化roups using flow cyto照農metry (FCM); (B) Cells外來 expressing an inducible 員年EGFP shRNA casset嗎用te showed a ~42% r會麗eduction in EGFP MFI upon IPTG inducti書場on. This observation was consist亮人ent across inducible 山民shRNA vectors carrying e算刀ither a puromycin算體 or blasticidin resistance gen生懂e. Inducible shRNA vector愛了s expressing a non-targeting s藍技cramble shRNA had no effec厭鐘t on EGFP MFI u作開pon IPTG induction.鐵道 Moreover, in cells 畫明transduced with an inducible shRNA 信數vector lacking the Lac做見I repressor, the induction function of銀草 the vector was lost and EGFP ex算工pression was consti女雪tutively inhibited by火黃 the EGFP shRNA both with and without I說喝PTG induction.

Tight regulation and maxi身舞mal induction: The presence of two repeats of the La小子cO element with弟快in the U6 promoter along with an a分笑dditional LacO element prese一愛nt downstream of the shRNA enables ti計呢ght regulation of shRNA expressio河短n by minimizing backg動輛round promoter activity in錯藍 the absence of IPT問上G and maximizing gene silencing in林刀 the presence of IPTG.

Fast response time: IPTG induced gene silencing can b聽照e achieved as fast as 48 hours.

Higher efficiency: Can achieve higher and more dy頻信namic knockdown than com金筆pared to a Tet-based inducible kn輛話ockdown system.&舊玩nbsp;

High viral titer: Our vector can be packaged in議商to high-titer virus (>10⁹ TU/ml when virus is ob請放tained through our virus packa下鐘ging service). At thi跳來s viral titer, trans舞就duction efficiency for culture林歌d mammalian cell如站s can approach 100% when a大南n adequate amount o制南f viral supernatant好那 is used.

Very broad tropism: 視哥;Our lentiviral packaging s就舊ystem adds the VSV-G envelo的放p protein to the viral surface. This p是森rotein has broad tropism. As a 業書result, cells from all com請子monly used mammalian species (an還拿d even some non-mam這錯malian species) can be transduced. Fur車友thermore, almost any mam樹請malian cell type can be tr慢我ansduced (e.g. dividing cells and non-我區dividing cells, primary cells and es信吧tablished cell lines, stem cells and 通雪differentiated cells, adheren影議t cells and non-adherent ce費制lls). Neurons, which are often i子關mpervious to conventional transfe工問ction, can be readily transduced by 新黑our lentiviral vector. Len慢火tiviral vectors packaged wit銀綠h our system have broade南費r tropism than ade喝道noviral vectors (which have low trans老我duction efficie哥呢ncy for some cell types了車) or MMLV retroviral vectors (whic熱醫h have difficult火算y transducing non-dividing cell喝還s).

Relative uniform章火ity of vector delivery:&n近跳bsp;Generally, viral t看些ransduction can deliver vector司視s into cells in 要工a relatively uniform mann們會er. In contrast, conventional transfect公美ion of plasmid vectors can be哥見 highly non-unifo亮女rm, with some cells recei物黑ving a lot of copies while other 這雪cells receiving few copies or n放西one.

Effectiveness in vitro and in 和如vivo: Lentiviral vector systems can be森算 used effectively in cultu照長red cells and in live animals.

Safety: The safety of our vector is ensured b業知y two features. One is the partiti現嗎on of genes required for v刀喝iral packaging and transduct很城ion into several helper plasmids;上女 the other is self-inactivation of th文些e promoter activity in t水林he 5' LTR upon vector村廠 integration. As a result, it女飛 is essentially imp呢小ossible for repl那美ication competent virus to 是老emerge during packaging and tran跳場sduction. The heal是水th risk of working with o在作ur vector is therefore min了睡imal.

Technical complexity低技: The use of lentiviral ve舊習ctors requires th新國e production of live virus in pac通區kaging cells followed by the能務 measurement of viral tit現制er. These procedures are technical d校兵emanding and time co我報nsuming relative to 費短conventional plasmid transfection.

RSV promoter: Rous sarcoma virus promoter問著. It drives transcription of viral RNA 謝飛in packaging cells. This RNA is then p厭化ackaged into live virus.來友

5' LTR-ΔU3: A deleted version of the 長但HIV-1 5' long terminal repeat. In wil空公dtype lentivirus生鐵, 5' LTR and 3' L西我TR are essentially identical i器看n sequence. They r房少eside on two ends 這到of the viral genome 鐘師and point in the same direct聽區ion. Upon viral integration長睡, the 3' LTR sequence is co老懂pied onto the 5'兒錯 LTR. The LTRs carry bo村物th promoter and pol站拿yadenylation functio學兒n, such that in wildtype virus, t路身he 5' LTR acts as a pr子道omoter to drive the transcription of th女著e viral genome, while 笑制the 3' LTR acts as a polyade術睡nylation signal to term理他inate the upstream transcript. On our v樂就ector, Δ5' LTR is delete冷空d for a region that is required 飛但for the LTR's prom北吧oter activity normally f子東acilitated by the viral tran費河scription factor信紙 Tat. This does not affec明業t the production of viral RNA durin國就g packaging because the promoter fun歌冷ction is supplemented by the RSV prom飛木oter engineered ups我冷tream of Δ5' LTR.

Ψ: HIV-1 packaging signal required for the關來 packaging of viral 吧短RNA into virus.

RRE: HIV-1 Rev response element. It allows學信 the nuclear export of viral RNA by the妹了 viral Rev protein dur山為ing viral packaging.

cPPT: HIV-1 Central polypurine tr了房act. It creates a "DNA flap" that incre多鐘ases nuclear importatio務訊n of the viral genome durin相公g target cell infecti是農on. This improves vector integrati土裡on into the host genome, resulting i朋件n higher transduction efficienc河店y.

U6/2xLacO: Modified human U6 small nuclear 1 著水promoter containing two rep友船eats of the lac operator sequenc那話e. Pol III promoter which suppress也哥es small RNA expression in the pre信匠sence of LacI.

Sense, Antisense: 冷火;These sequences are derived from your近章 target sequence and are transcribe制放d to form the stem portio雜去n of the “hairpin” structure of the都草 shRNA.

Loop: This optimized seque外答nce is transcribed to form the 什土loop portion of 個近the shRNA “hairpin” structure.

Terminator: Terminates transcription of分見 the shRNA.

LacO: Lac operator. In the ab厭行sence of IPTG, i服也t is bound by lac repressor (LacI), lea秒能ding to transcriptiona秒舊l repression of 低村the upstream shRNA.通就 In the presence of IPTG, LacI can no 商火longer bind to LacO, thus allow鄉去ing upstream shRNA to b用做e transcribed.

hPGK promoter: 林作;Human phosphoglycerate事腦 kinase 1 gene promoter. It driv歌頻es the ubiquitous expre遠跳ssion of the downstream 謝月ORF.

Regulatory protein: 畫關Lac repressor (LacI) and a drug se票小lection gene (such 很山as puromycin resistance 技唱gene) linked by T2A linker. Allow和計s cells to express快睡 LacI protein and be resistant to the通光 corresponding drug. In紅商 the absence of IPTG山短, LacI binds to LacO t動又o repress transcriptio腦銀n of downstream genes or small 答日RNAs. In the presenc空喝e of IPTG, LacI undergo對子es a conformational change and is no 白風longer able to bind to LacO, thus中黃 allowing downstream genes or s唱計mall RNAs to be 玩分transcribed.

WPRE: Woodchuck hepatitis virus posttranscrip身綠tional regulator市的y element. It enhances viral RNA sta體雜bility in packaging房雨 cells, leading to higher titer of 土錯packaged virus.

3' LTR-ΔU3: A truncated version of the HI票和V-1 3' long terminal repe喝樂at that deletes the U3 reg市業ion. This leads to the self-ina大熱ctivation of the pro現算moter activity of the 5' LTR up兒區on viral vector integration into the照會 host genome (due to the fact that 3' L廠也TR is copied onto 5' LTR durin但空g viral integration). The polyadenylat吃電ion signal contained in 鐘機ΔU3/3' LTR serves to termin畫師ates all upstream tra靜舞nscripts produced both during vira長機l packaging and afte舞筆r viral integration into the host gen東白ome.

SV40 early pA: Simian virus 40動這 early polyadenylation signal. It furt地理her facilitates transcripti老對onal termination after the 3' LTR du報林ring viral RNA transcript土微ion during packaging. This elevates 紅紅the level of functional vir唱如al RNA in packaging謝長 cells, thus improving viral titer.

Ampicillin:&nbs公花p;Ampicillin resistance gene. It al志劇lows the plasmid to be ma拿店intained by ampicillin selec北還tion in E. coli.

pUC ori: pUC origin of replication. Plasmids c件分arrying this origin exist分民 in high copy numbers車北 in E. coli.