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MSCV Retrovirus Non-Coding RNA E家海xpression Vector
概況
The MSCV (Murine stem c制裡ell virus) retroviral vector 就好system is a hig紅兵hly efficient vir歌民al vehicle for permanentl靜紙y introducing non-coding RNAs of in外文terest in embryonic stem (ES) cells, em黑光bryonal carcinoma (EC)裡兵 cells and hematopoietic stem (HS) 體動cells along with severa通廠l other mammalian cell lines.們音 Non-coding RNAs include a wi店老de variety of short (&l章輛t;30 nucleotides) and lon吧身g (>200 nucleotides) fu作花nctional RNA molecules suc子水h as microRNAs (miRNAs), small i靜頻nterfering RNAs (siRNAs),光農 piwi-interacting RNAs 章年(piRNAs), small nuclear RNAs (海術snRNAs), small nucleolar RNAs (snoRNA機票s), large inter木玩genic non-coding RNAs (l都工incRNAs), intronic long no內小n-coding RNAs (intronic lncRNAs)輛喝, natural antisense tran短麗scripts (NATs), enhan時通cer RNAs (eRNAs) and promote科靜r-associated RNAs (PARs), n訊還one of which are translated int務哥o proteins, howeve紅說r have been found to嗎討 play important roles in many cellular器明 processes such as DNA rep站行lication, epigenetic regulat微雪ion, transcriptional紅鄉 and post-transcriptional regula兵物tion and translation regulation.
The MSCV retrovirus non上照-coding RNA expression v站風ector uses the ubiquitous p書秒romoter function in the 5’ LTR (空爸long terminal repeat) 海地of the MSCV retroviral gen子朋ome to drive the expressio日照n of the user-selected non-coding R跳有NA gene, which is mediated by RNA polym視內erase ll-dependent tra頻綠nscription. For RNA polymerase ll-medi外亮ated transcription, 師船the start site is 雪習typically in the 3車雪’ region of the promoter while the ter事亮mination site is within the p場熱olyA signal sequence. A我些s a result, the trans們房cript generated from this vector do林雨es not correspond precisely to the sel森紅ected non-coding RNA ge鄉但ne, but contains some additional sequen光術ces both upstream and dow在上nstream.
An MSCV retroviral vec動習tor is first construct你刀ed as a plasmid in E. coli. The non-吃綠coding RNA of interest is如照 cloned between the two LTRs during ve笑市ctor construction. It is then tr海河ansfected into packaging 費多cells along with sev關拿eral helper plasmids. Inside the pa黑為ckaging cells, vector DNA located betwe樂下en the two LTRs is弟資 transcribed into RNA, a窗術nd viral protein小市s expressed by the helper plasmids fur技訊ther package the RNA into吧睡 virus. Live virus is通畫 then released into the supern月了atant, which can be used to infe見裡ct target cells d用雜irectly or after concentration.
When the virus is added to 黑靜target cells, the RNA cargo is shuttl是睡ed into cells where it美中 is reverse transcribed into玩子 DNA and randomly integrated i林身n the host genome. Any gen醫間e(s) that were placed in-between t個信he two LTRs during vector cl女路oning are permanently i明就nserted into the host DNA 小女alongside the rest of費文 the viral genome.
By design, MSCV retroviral vecto開跳rs lack the genes required for vir飛月al packaging and transduction (t哥舞hese genes are carried by helper 頻樂plasmids or integrated int中算o packaging cells i票朋nstead). As a result, 討綠viruses produced from the vectors have 通遠the important safety feature短機 of being replication incompetent (數離meaning that they can農光 transduce target cell店海s but cannot replic事懂ate in them).
For further info少跳rmation about this vector system友關, please refer t來醫o the papers be自多low.
| References | Topic |
|---|---|
| Cell. 157:77 (2014) | Review on non-coding RN相西As |
| Front Genet. 6:2 (2015) | Review on functionality of non-codin年匠g RNAs |
| PLoS One. 8:e77070 (2013) | Retrovirus-mediated expression of l一學ong non-coding RNA |
| Exp Hematol. 31月老: 1007 (2003) | Review on retrovirus雪機-mediated gene expression |
| Gene Ther. 1: 136 (1994) | Development of the MSCV vector ser工遠ies |
亮點
Our vector is optimized for h去兵igh copy number replication in E. c慢訊oli, high-titer packaging報議 of live virus, efficient viral tran司線sduction of a wide range of cells inc就線luding ES, EC and 車從HS cells, efficient vecto裡暗r integration into the host genome, an舞得d high-level transgene expression可自.
優勢
Permanent integration of v校她ector DNA: Conventional trans雨在fection results子來 in almost enti厭中rely transient delivery of DNA into ho化鐵st cells due to我這 the loss of DNA over ti都短me. This problem is especially promi匠醫nent in rapidly dividin就新g cells. In cont站從rast, retroviral t劇海ransduction can deliver genes pe技自rmanently into h工慢ost cells due to i影窗ntegration of the viral去去 vector into the host genome.
Broad tropism: Our packaging system adds th藍很e VSV-G envelop protein 廠去to the viral surface. Th空術is protein has broad tropism. A鐘費s a result, cells from com刀吃monly used mammalian species such as h家店uman, mouse and rat can be t通外ransduced. Addi土廠tionally, the presence of 妹西a strategically designed 5’LTR de答慢rived from the PCMV v化生irus in the MSCV retroviral vec姐腦tor helps to achieve hi亮笑gh level expression 什站of target genes in ES, 生子EC and HS cells. This offers a distinct章務 advantage over MML白金V retroviral vectors which s道技how a restricted expression of t那說arget genes in ES and EC cells. Ho到錢wever, our MSCV vec金會tor has difficulty transducing non-如司dividing cells (see disadv風我antages below).
Large cargo space: The genome for th間司e MSCV retroviral vector is ~8 kb. In小機 our vector, the components necessary船木 for viral packaging and t暗路ransduction occupy ~1.9 kb, which leav得技es ~6.1 kb to accommodate the user'機不s DNA of interest. 雪麗Because our vector is designed 國子for the insertion of only a non-c身嗎oding RNA sequence, this cargo space is畫妹 sufficient for most 爸很applications.
High-level expression: The 5' LTR contains a strong ubiquito離購us promoter that drives hig風人h-level expression of the user's non山北-coding RNA of inte雨關rest.
Relative uniformity of gene deliv那海ery: Generally, viral 鐵中transduction can deliver v答體ectors into cells i放厭n a relatively uniform manner. In cont習北rast, conventional 做放transfection of plasmid vect市高ors can be highly non村年-uniform, with some cel海紅ls receiving a lot of歌房 copies while other cells rece空看iving few copies or n線公one.
Effectiveness in vitro and in vivo: While our vector is mostly是在 used for in vitro transduction of cult通樂ured cells, it can also be used姐能 to transduce cells in live an區個imals.
Safety: The safety of our ve輛窗ctor is ensured by pa多什rtitioning genes required for vir嗎算al packaging and tr銀內ansduction into several help鄉美er plasmids or integrating them 作小into packaging cells. As a result,員身 live virus produce通飛d from our vector is replication incomp數個etent.
不足之處
Dependence on 5' 讀做LTR promoter: Expression of the non-c上照oding RNA in our vecto兵車r is driven by t新但he ubiquitous p道東romoter function in the 5' LTR. Thi中能s is a distinct di公可sadvantage as compar子又ed to our lentiviral vectors which al資通low the user to p聽木ut in their own promoter to d子文rive their non-codin書影g RNA of interest.
Moderate viral titer房你: Viral titer from文化 our vector reaches ~107 TU/ml in the上草 supernatant of pa體司ckaging cells without further明男 concentration. This is about 白美an order of magnitude lowe時個r than our lentivira技票l vectors.
Difficulty transduc金多ing non-dividing cells: MSCV has difficulty transducing n不多on-dividing cells.
Technical complexity: The use of MSCV retrovira麗兵l vectors requires the些區 production of live virus in packagi道厭ng cells followed 相通by the measurement of vir海花al titer. These procedures are t一司echnically demanding and time con農計suming relative to 時飛conventional plasmid 好兒transfection.
關鍵元件
MSCV 5’ LTR: 5' long terminal re你見peat from PCC4-cell-passaged myeloprol有從iferative sarcoma virus (PCMV).見術 The LTRs carry拿到 both promoter a林務nd polyadenylation 紅到function, such th多來at the 5' LTR acts as a promote在些r to drive the transcription of the vir務相al genome, while t這頻he 3' LTR acts as a polyadeny慢知lation signal to terminate the upstrea船河m transcript. The 5’ LTR deriv民金ed from the PCMV retrovirus in the照你 MSCV vector has been strategically mod湖草ified to drive the transcriptional友費 activation of target genes i在資n pluripotent cell lines such as ES or現作 EC cells, unlike MMLV retrovi綠上ral vectors.
MSCV Ψ： Murine embryonic stem cell virus pac湖明kaging signal required for pac但錢kaging of viral RNA into virus.
Non-coding RNA: Your non-coding RN看木A of interest is placed here.線我 Its expression is dri關醫ven by ubiquitous promoter func市西tion in the 5’ LTR.音商
MSCV 3’ LTR: 3' long terminal repeat from PCMV. It a熱件llows packaging of vir志司al RNA into virus and fa是醫cilitates transcription term工站ination and mRNA polyadenyla懂站tion in ES cells and o暗唱ther cell types.
pUC ori: pUC origin of re民費plication. Plasmids carrying thi刀舞s origin exist in high copy低鐘 numbers in E. coli.
Ampicillin: Ampicillin resistance gene. It al水船lows the plasmid to be maintai南關ned by ampicillin selection in 服費E. coli.