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Lentivirus Non-Coding RNA Expression V內妹ector
概述
The lentivirus non-coding RNA exp銀呢ression vector is a highly effici亮拍ent vehicle for 分又permanently introducing non-co務議ding RNAs of interest in ma書坐mmalian cells. Non-coding員東 RNAs include a wide variety of short 農知(<30 nucleotides) and long (>200 員站nucleotides) functional RNA molecules s山議uch as micro RNAs (miRNAs), small int鐵信erfering RNAs (siRNAs),車這 piwi-interacting RNAs (piRN信頻As), small nuclear RNAs (snR多人NAs), small nucleolar RNAs (snoRNAs可好), large intergenic non-coding RNAs (l文得incRNAs), intronic long non-coding RNA藍木s (intronic lncRNAs)用讀, natural antisense transcript樂通s (NATs), enhancer RNAs (eRN購鐘As) and promoter-associat國學ed RNAs (PARs), none o行們f which are translated into proteins, 司跳however have bee吃學n found to play important rol但為es in many cellular proces一讀ses such as DNA replic志對ation, epigenetic r制高egulation, transcriptional and見行 post-transcriptional r畫事egulation and translation regulati中如on.
The lentivirus non站我-coding RNA expression vect票物or uses an 城老;RNA polymerase II promoter to drive件樹 the expression of the user-sel線了ected non-coding RN拍器A gene. This allows 黑笑the use of tissue-sp湖生ecific, inducible, or variable-s木書trength promoters, enabling a空城 variety of experiment刀關al applications. Fo工些r RNA polymerase II-街如mediated transcr制到iption, the start site is typ謝醫ically in the 3' regio作不n of the promoter while the termi媽但nation site is within t飛見he downstream polyA 大視signal sequence. As a result, the tr歌城anscript generated from this vector d日數oes not correspond precisely to輛光 the selected non-coding RNA 拿錢gene, but contains some 草高additional sequ厭會ences both upstream an看慢d downstream. For th森那e lentiviral vector, int都長ernal polyadeny場月lation signal is 唱低not suggested to be placed between 數電the LTRs for each individu新劇al expression cassette, as th城劇is would inhibit virus p很秒ackaging. Therefore, sequenc是冷e downstream of t算件he non-coding RNA till 3' LTR will be t從鐵ranscribed along with the non-codin舊商g RNA which may affect the民亮 non-coding RNA funct看快ion.
The lentivirus non-coding 路輛RNA expression vector is fir開窗st constructed as a plasmid i北長n E. coli. The non-coding RNA of 技大interest along with a user-selected 見報promoter is cloned between the two l拿腦ong terminal repeats (LTRs) during vec靜去tor construction. 資了The vector is t會呢hen transfected間章 into packaging cells 說資along with several helper plasmi微不ds. Inside the packaging cells, vector 哥草DNA located between好機 the LTRs is transcribed into RNA, an笑空d viral proteins expressed by the hel關湖per plasmids further packa街長ge the RNA into virus. Live virus 機兵is then released into樹妹 the supernatant, which can be used to 嗎船infect target cells direct綠討ly or after concentration.
When the virus is added t市地o target cells, the日子 RNA cargo is shuttled into cells whe畫妹re it is reverse transcribed into到黑 DNA and randomly in多報tegrated into the窗還 host genome. The non-coding自做 RNA sequence placed in-betw劇這een the two LTRs during v窗頻ector construction is permanently inser問資ted into host DNA alongside the r小上est of viral genome.
By design, lentiviral vectors lack我資 the genes required for viral司拍 packaging and transduction (these事文 genes are instead carried by helper p章美lasmids used duri會暗ng virus packaging). As a做火 result, virus produced from lentiviral鐵暗 vectors has the important safety fea很房ture of being replication incompe呢離tent (meaning that they can transduce女拿 target cells b火書ut cannot replicate in長很 them).
For further information頻有 about this vector s金玩ystem, please refer to the paper爸到s below.
| References | Topic |
|---|---|
| Cell. 157:77 (2014) | Review on 制線non-coding RNAs |
| Front Genet. 6:2 (2015) | Review on functionality of non-codin們小g RNAs |
| Int J Oncol. 48:1509 (2016) | Lentivirus-mediated expression of lo到但ng non-coding RNA |
| J Virol. 72:8463 (1998) | The 3rd generation lenti些高virus vectors |
| J Virol. 72:9873 (199數匠8) | Self-inactivating lentivirus vectors |
| Nat Protoc. 1:24是靜1 (2006) | Production and purification of lent懂件iviral vectors |
亮點
The lentivirus non-coding RNA 能報expression vector is derive舞如d from the third-gen線得eration lentiviral vector s來務ystem. It is optimized 喝民for high copy number replication i空畫n E. coli, high用河-titer packaging of live vi時歌rus, efficient viral tr知亮ansduction of a wide呢師 range of cells, efficient vector integ信中ration into the聽聽 host genome, and high-level transgene到場 expression.
優勢
Permanent integr票訊ation of vector DNA: Conventional transfection畫快 results in almost土習 entirely transien近紅t delivery of DNA 影友into host cells due厭些 to the loss of DNA over time. This 吧河problem is especially promi們請nent in rapidly divi水年ding cells. In contr放花ast, lentiviral transduction can 開機deliver non-coding RNAs permanen這相tly into host cell家書s due to the integration of the viral 員相vector into the ho多美st genome.
High viral titer: Our lentivira信還l vector can be packaged into hig國路h titer virus. When lenti說窗virus is obtained through our v西朋irus packaging service, 業刀titer can reach >109 transducing unit p子中er ml (TU/ml). At this tit照但er, transduction呢都 efficiency for cultured mammalian ce道美lls can approach 100% wh機到en an adequate amount of viral is use女討d.
Very broad tropi北做sm: Our packaging s時醫ystem adds the VSV-G envelop 大物protein to the viral surface. This prot飛笑ein has broad tropism. As a resul就票t, cells from all commonly 外人used mammalian sp外黃ecies (and even some 答見non-mammalian species) can b歌路e transduced. Furthermore, almost any 數林mammalian cell type can be transduced 讀動(e.g. dividing cells and嗎商 non-dividing cells, primary cells and個員 established cell lines, stem cells an書要d differentiated cell得長s, adherent cells and non-adhere個喝nt cells). Neurons, w商看hich are often i校工mpervious to conventiona畫遠l transfection, can be re畫離adily transduced by our lenti空離viral vector. Lentiviral vector制北s packaged with our sys事雨tem have broader輛男 tropism than adenovir行船al vectors (which have low t子資ransduction efficiency for som都做e cell types) or MMLV retroviral ve畫城ctors (which have difficulty transduci的近ng non-dividing 資多cells).
Customizable internal promot站們er: Our vector is designed to self-in理理activate the promoter activity in it員事s 5' LTR upon integration金笑 into the genome. As a result, user議紅s can put in th都飛eir own promoter to drive their 長風non-coding RNA of interest within th裡工e vector. This is a distinct advantag舊好e over our MMLV re數外trovirus vectors, which微海 rely on the pr熱服omoter function of 5' LTR to drive 校白the ubiquitous expression 信民of the gene of interest.
Relative uniformity of gene d煙們elivery: Generally, viral transduction can d煙看eliver vectors into 線業cells in a relativel和線y uniform manner. In contrast, convent麗對ional transfection of plasmid vectors 房機can be highly non-uniform, with so月又me cells receiving a lot頻房 of copies while other cells rec窗校eiving few copies or none.
Effectiveness in vitro and in vivo:舞得 While our vector生愛 is mostly used for in vitro t身美ransduction of cultured cells, it can 做畫also be used to 影音transduce cells in live ani外小mals.
Safety: The safety of our 知劇vector is ensured by two fe喝日atures. One is the partiti公雜on of genes required f月西or viral packaging and tr裡身ansduction into several helper plasmi視票ds; the other is self-inactivation o唱車f the promoter activity in如低 the 5' LTR upon vector integrat著中ion. As a result, it is essentially imp文村ossible for replication comp秒章etent virus to emerge during packaging跳歌 and transduction. The health 森林risk of working with our vector is ther門都efore minimal.
不足之處
Limited cargo space: The wildtype lentiviral genome is 鐵呢~9.2 kb. In our vector, the compo習雪nents necessary for viral packaging市大 and transduction occup但近y ~2.8 kb, which leaves ~6.4 kb t服火o accommodate the user's那們 DNA of interest. W河裡hen the vector goe男聽s beyond this size limit, vir跳廠al titer can be severely reduced. Our低區 vector is routin訊厭ely used for in錯關serting several functional e舞中lements besides the non-coding RN湖厭A of interest, such快上 as promoter and d東藍rug resistance. A large non-coding R老鐘NA plus these additiona醫民l elements could excee可高d 6.4 kb, and the r要船esult could be compr城腦omised viral production.
Technical complexity和男: The use of lentiviral vectors require窗飛s the production of l腦日ive virus in pa去暗ckaging cells followed by the measure到拍ment of viral titer. Thes事鐘e procedures are techn商國ically demandin近黑g and time consuming r筆時elative to conven又媽tional plasmid transfection.
載體關鍵元件
RSV promoter: Rous sarcoma virus promo金金ter. It drives tra化吃nscription of viral RNA in packagi工都ng cells. This RNA is then packag拿電ed into live virus.
5' LTR-ΔU3: A deleted version of t會拿he HIV-1 5' long terminal repeat. In大小 wildtype lentivi能化rus, 5' LTR and 3' LTR are essential做冷ly identical in sequence. They reside 女煙on two ends of the viral電物 genome and point in the same dir司民ection. Upon viral in風他tegration, the 3' LTR sequence 員明is copied onto the 5' LTR. The錯她 LTRs carry both p店計romoter and poly照山adenylation function, such tha機低t in wildtype virus, the 5了但' LTR acts as a promoter to drive the 服銀transcription of t白睡he viral genome, while the 3' 你東LTR acts as a polyade銀她nylation signal to termin著到ate the upstream tr線費anscript. On our vector, Δ5' LTR腦輛 is deleted for a region 話業that is required for the LTR's promot道山er activity normally facilitated b公新y the viral transcripti通黑on factor Tat. This does not a算坐ffect the productio民用n of viral RNA duri爸朋ng packaging because the p地喝romoter function is supp匠作lemented by the RSV promote人內r engineered ups公又tream of Δ5' LTR.
Ψ: HIV-1 packaging s白務ignal required fo船東r the packaging of viral RNA int技大o virus.
RRE: HIV-1 Rev response element. 和得It allows the n話黃uclear export of viral RNA 男友by the viral Rev protein during viral p放山ackaging.
cPPT: HIV-1 Central polypurine為師 tract. It creates a "DNA flap" 跳明that increases nuclear importa文個tion of the viral genome during targe小外t cell infectio問議n. This improves vector integrati光北on into the host genome, resulting in費電 higher transduction 可森efficiency.
Promoter: The promoter dr可舞iving your non-coding RNA&nbs土了p;of interest is placed here.
Non-coding RNA: The non-coding RNA of your i來說nterest is placed here.
WPRE: Woodchuck hepatitis vir城嗎us posttranscriptional regulatory elem白費ent. It enhances viral RN技為A stability in packaging視相 cells, leading to higher titer of p那話ackaged virus.
mPGK promoter:&nb近筆sp;Mouse phosphoglycerate kinase 1 gene p我子romoter. It dri習就ves the ubiquitous expression 林煙the downstream marker gene.
Marker: A drug selection gene (su歌事ch as neomycin resistance), a低水 visually detectable g就了ene (such as EGFP), or 機愛a dual-reporter gene 得事(such as EGFP/Neo). This 員子allows cells transduced wit錢廠h the vector to be sele制火cted and/or visua村們lized.
3' LTR-ΔU3: A truncated version的門 of the HIV-1 3' long termin多錯al repeat that deletes th男從e U3 region. This leads to the self中服-inactivation o少藍f the promoter activ我關ity of the 5' LTR 電相upon viral vector integrati嗎匠on into the host geno離遠me (due to the fact that 3' LTR i匠呢s copied onto 5' LTR during viral inte費了gration). The polyaden上年ylation signal cont票去ained in ΔU3/3' LTR serves to terminate車城s all upstream tran些年scripts produced both during v車兵iral packaging and after v女讀iral integration i上店nto the host genom技書e.
SV40 early pA: Simian virus 40 early兒見 polyadenylation signal. It further朋低 facilitates transcr線很iptional termination after the 3' LTR d醫章uring viral RNA transcription durin是煙g packaging. This elevates the level o玩是f functional viral RNA 校近in packaging cells, thus impro音她ving viral titer.
Ampicillin: Ampicillin resistance gene. I請紅t allows the plasm畫銀id to be maintained by雪雪 ampicillin select外友ion in E. coli.
pUC ori: pUC origin of 去現replication. Plasmids carrying this ori他少gin exist in hi風西gh copy numbers in E. coli.