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哺乳動物ncRNA表達自失活型MMLV載體
概述
The MMLV retrov睡慢iral vector system is an efficient樂時 vehicle for intr議水oducing non-coding RNAs p姐到ermanently into mamm城懂alian cells. Non-coding RNAs 森路include a wide 影金variety of short (<30 光錢nucleotides) and long (&g腦城t;200 nucleotides) functional RNA mole討技cules such as micro RNAs 器行(miRNAs), small interferi動相ng RNAs (siRNAs), piwi熱讀-interacting RNAs (piRNAs), smal土了l nuclear RNAs (snRNAs), small nuc都坐leolar RNAs (snoRNAs), large 窗理intergenic non-coding RNAs (l月刀incRNAs), intronic long國說 non-coding RNAs (intronic lncRNAs科亮), natural antise是友nse transcripts (NATs), e訊多nhancer RNAs (eRNAs) and promot筆下er-associated RNAs (PARs), none o放遠f which are translated into pro通白teins, however have been found t水短o play important近村 roles in many cellular p東林rocesses such as DNA rep樹線lication, epigenetic regulation, t遠家ranscriptional and post-transcriptio劇物nal regulation and translation 相章regulation.
MMLV, a retroviral ve影還ctor derived from 低歌Moloney murine leukemia virus, is a友腦 plus-strand linear RNA virus that exh風村ibits efficient genomic integr錢呢ation. While our wildtype MMLV re北近trovirus expression vector util南林izes the ubiquit兒科ous promoter function in the 5' long地綠 terminal repeat (LTR) of wildtype M些東MLV genome for 站錯driving expression of the non-coding RN這紅A, the self-inactivati門志ng MMLV retrovirus expression 子朋vector allows users to select any pro山日moter of their choice for driving no拍我n-coding RNA expression.大文 This is achiev妹了ed by the deletion 現跳of the U3 region i文暗n the MMLV 3’ LTR whi北村ch self-inactivates the promot吃很er activity in 見什the 5' LTR by a copying mechanism去女 during viral genome integrati制年on. This not only訊錢 provides users with做錢 the flexibility to ad南聽d their promoter of choice for坐路 driving non-coding RNA歌讀 expression but also elim學微inates the risk 森知of oncogenic activation of a玩農djacent genes upon vector integ東個ration, thereby enabling such vector開南s to have a higher safety profile c森男ompared to wildtyp章這e MMLV vectors.
A self-inactivating MMLV retroviral ve問麗ctor is first constructed as a plasmid南舊 in E. coli. It船新 is then transfected into packa的美ging cells along with s我笑everal helper plasmids. Insi靜熱de the packaging cells, vecto到北r DNA located between the LT業近Rs is transcribed into RN店高A, and viral proteins expressed by 快照the helper plasmids 花個further package the RNA into 舞作virus. Live virus is then r農亮eleased into the supernatant, which c我妹an be used to infect target些但 cells directly or afte討務r concentration.
When the virus is added to target ce都話lls, the RNA cargo is shutt訊得led into cells where it 用村is reverse transcribed into看短 DNA and randomly in睡銀tegrated in the host genom訊好e. The non-coding RNA sequence th作新at was placed in新動-between the two LTRs during vec金拍tor constructio廠喝n is permanently 麗輛inserted into host 光線DNA alongside the rest of v物看iral genome.
By design, self-inactivating MMLV re刀麗troviral vectors lack the genes requir車著ed for viral pa小什ckaging and transduction (these g和老enes are carried by helper p西錢lasmids or integrated into packagi件懂ng cells instead). As a result, vi秒器ruses produced from the煙行se vectors have the impor著通tant safety fea亮答ture of being replication incompetent 船音(meaning that th訊聽ey can transduce target cel物答ls but cannot replicate in the看我m).
For further informat做木ion about this 水國vector system, please refer to the pape綠金rs below.
| References | Topic |
|---|---|
| Cell. 157:77 (2014) | Review on non-coding RNAs |
| Front Genet. 6:2 (2015) | Review on functionality of n得購on-coding RNAs |
| PLoS One. 8:e77070 (2013) | Retrovirus-mediated expres關費sion of long non-coding RN能章A |
| J Virol. 61:1639 (198器車7) | Extended packaging signal increases t些會he titer of MMLV vectors |
| Gene Ther. 7:1063 (錯鐘2000) | Tropism of MMLV vecto麗時rs depends on packagin拿子g cell lines |
| Proc Natl Acad Sci USA. 83:3194媽影 (1986) | Designing of se電劇lf-inactivating retroviral vec校農tors for gene trans中林fer into mammalian ce可很lls |
亮點
The SIN MMLV retrovir現照us non-coding RNA expression vecto分下r is optimized for high c東放opy number replication in E. coli在又, high-titer packaging of 算拍live virus, efficient viral tra山快nsduction of a wid和從e range of cells, effi微冷cient vector integration in內報to the host genome, and high-level e內機xpression.
優勢
Permanent integration來下 of vector DNA: Conventional transfection results 得河in almost entirely transient delivery站雪 of DNA into hos但美t cells due to th暗的e loss of DNA over time. This答電 problem is esp冷什ecially prominent in rapidly divid爸廠ing cells. In contrast, re鐵數troviral transductio喝少n can deliver non-長舞coding RNAs permanently into host ce東員lls due to integration of the v地明iral vector into the host genome.
Broad tropism: Our packaging system adds the V國舞SV-G envelop protein to著人 the viral surface. This pr雨體otein has broad tropism. As a放姐 result, cells from a低放ll commonly used mam讀鐵malian species such as human,如街 mouse and rat can be t工村ransduced. Furthermore, many specif鐵舞ic cell types can 歌船be transduced, 時醫though our vector has d畫子ifficulty transducing non-dividin藍請g cells (see disadvantages below).
Customizable internal promoter: 業拍;Our vector is designed人吧 to self-inactivate the promoter activi制東ty in its 5' LTR upon integration i老答nto the genome. As a resul藍樹t, users can put in their own promoter 睡們to drive their non-coding RNA with學書in the vector. This is時慢 a distinct advantage over門窗 our wildtype MMLV 高腦retrovirus vectors, w東湖hich rely on the promoter fu秒的nction of 5' LTR to drive the ubiquito空請us expression.
Relative uniformity o雨窗f delivery: Generally, viral transduc拍件tion can deliver vectors into cel什志ls in a relatively unifor在件m manner. In contrast, conventio明多nal transfection 聽動of plasmid vectors can be highly non-照費uniform, with some ce什輛lls receiving a lot of copies whi些村le other cells re女子ceiving few copies or none.
Effectiveness in 農但vitro and in vivo:&nb少舞sp;While our vecto懂笑r is mostly used for in vitro tran海這sduction of culture校作d cells, it can 計高also be used to transduce cells國嗎 in live animals.
Safety: The safety of our vector is ensured 路麗by two features. One is the part微好itioning of genes required for 村子viral packaging and transduction吧相 into several helper plasmids; the 為朋other is self-inactivation of the pr新讀omoter activity電子 in the 5' LTR upon vector 說少integration. As a result, it新弟 is essentially impossib街是le for replication competent virus to 林現emerge during packaging an山多d transduction. The health risk如喝 of working with 媽弟our vector is therefore minimal.
不足之處
Moderate viral tite暗小r: Viral titer from our廠那 vector reaches ~107 TU/ml in the super器時natant of packaging 新訊cells without fur低文ther concentration. T家還his is about an order of 吧市magnitude lower than our lentiviral 科學vectors.
More limited carg從水o space than wildt國筆ype MMLV: The MMLV retroviral genome i報月s ~8.3 kb. In our 話懂vector, the com著短ponents necessa中來ry for viral packaging and 我件transduction occupy 聽空~2.6-3 kb, which老還 leaves only ~5讀從.3-5.7 kb to accommodate the u個化ser's DNA of interest, including bo離知th the non-coding RNA 來為and the promoter.
Difficulty transducing non-div有哥iding cells: 輛房;Our vector has difficult也書y transducing non-dividing cells.
Technical compl家慢exity: The use of MMLV retroviral vecto志嗎rs requires the production of li身頻ve virus in packaging cells 醫訊followed by the measurement 村業of viral titer. Thes內哥e procedures are technically dema錢術nding and time consuming re開亮lative to conventional pla新著smid transfection.
載體關鍵元件
CMV promoter: Human cytomegalovirus immediate earl木遠y promoter. It drives transcri友新ption of viral RNA in pa得坐ckaging cells. This RNA is then p到地ackaged into live virus.
MMLV 5' LTR-ΔU3:&筆就nbsp;A deleted version of the MMLV 技拿retrovirus 5' long terminal repeat. I事看n wildtype MMLV retroviru家學s, 5' LTR and 3' LTR are essentially 弟我identical in sequence. They reside o南內n two ends of the viral gen照間ome and point in the same dire站窗ction. Upon viral integration, the 不多3' LTR sequence is copied ont個筆o the 5' LTR. The LTRs carry女兵 both promoter and polyadeny樂費lation function, such that the熱路 5' LTR acts as a promot呢明er to drive the transcription of the 機船viral genome, while the 3' LTR acts as外秒 a polyadenylation signal to te也玩rminate the upstream tran身南script. On our vector, MM術輛LV 5' LTR-ΔU3 is deleted for a 兒湖region that is required for the說裡 LTR's promoter activity. This 店習does not affect the 時聽production of viral RNA d我章uring packaging because the promoter f議了unction is suppleme海上nted by the CMV promoter engineered ups弟火tream of Δ5' LTR.
Ψ plus pack2: MMLV retrovirus packaging sign黃銀al required for 腦一the packaging of vira問北l RNA into virus.
Promoter: The promoter driving expression 煙吃of your non-coding R房畫NA is placed here.
Non-coding RNA: The non-coding RNA o小刀f your interest is 那我placed here.
WPRE: Woodchuck hepatitis virus pos行近ttranscriptional regulatory element. I鐘事t enhances viral RNA sta吃唱bility in packaging cells, leadin也湖g to higher titer of中些 packaged virus.
MMLV 3' LTR-ΔU3: A truncated區請 version of the MMLV們亮 retrovirus 3' long te議個rminal repeat. Th喝河is leads to the self-inacti開兒vation of the promoter activi到匠ty of the 5' LTR數就 upon viral vector integration into視歌 the host genome (due to the fact that 鐵雜3' LTR is copied onto 5' LTR 子技during viral integr自開ation). The polyadenylation sign如兒al contained in MMLV 3' LT坐章R-ΔU3 serves to terminate all 事兒upstream transcripts 外劇produced both durin票銀g viral packaging and 業數after viral integration 友車into the host genome.
SV40 late pA: Simian vir靜務us 40 late polyadeny腦很lation signal. It有地 further facilitates transcriptio友個nal termination afte做有r the 3' LTR during packaging. This e老能levates the level of functional viral R風輛NA in packaging 煙服cells, thus improving viral titer.
pUC ori: pUC origin of replica低是tion. Plasmids carrying this origin大計 exist in high copy numbers in E. co用海li.
Ampicillin: Ampicillin resistance gene. It allo音開ws the plasmid to be maintained 一商by ampicillin selection in E. coli.
Mammalian ncRNA E匠刀xpression Self-Inactiv制票ating MMLV Vector
概述
The MMLV retroviral vector system is an紙黑 efficient vehicle for introducing non-腦頻coding RNAs permanently 門腦into mammalian cells國著. Non-coding RNAs include a wide va請村riety of short (&我玩lt;30 nucleotides海是) and long (>200 nuc遠區leotides) functional RNA molecul為畫es such as micro RN照船As (miRNAs), small interfering RNAs但知 (siRNAs), piwi-inter花放acting RNAs (piRNAs), sm還事all nuclear RNAs (snRNA拍來s), small nucleola呢什r RNAs (snoRNAs), large站匠 intergenic non-coding RNAs (linc哥長RNAs), intronic long non-coding RN水資As (intronic lncRNAs),看草 natural antisense tra算習nscripts (NATs), enhancer RNAs (e鄉年RNAs) and promoter-associate森懂d RNAs (PARs), 林化none of which are紅那 translated into proteins, however ha歌她ve been found to play important r相空oles in many cel是謝lular processes 票玩such as DNA replication, epigenetic 長離regulation, tra白員nscriptional and post-transcriptiona站朋l regulation and t來我ranslation regulat樂雜ion.
MMLV, a retroviral vect門唱or derived from Moloney murine 章裡leukemia virus, is a plus-strand lin嗎紅ear RNA virus that exhibits 熱視efficient genomic int不制egration. While our wildtype MML光校V retrovirus expression vector u數通tilizes the ubiquitous promoter functio裡看n in the 5' long ter金紙minal repeat (LTR) of wildtype MML謝照V genome for driving expressio科美n of the non-coding RNA, the自件 self-inactivatin服小g MMLV retrovirus expr線制ession vector allows u器技sers to select any promoter費腦 of their choice 村呢for driving non-coding RNA expression.但好 This is achieved by 視場the deletion of the U3 如區region in the MMLV 3’ LTR wh站視ich self-inactivates the promot城服er activity in the 5' LTR by a copy河場ing mechanism during vir離不al genome integration. This not only pr山銀ovides users with th技一e flexibility to add their promoter of很對 choice for driving non-codin機微g RNA expression but also eli匠化minates the risk of oncogenic acti聽匠vation of adjacent genes upon ve公山ctor integration, thereby enabling光一 such vectors to have a higher s舊拿afety profile compared to wildtype MM機在LV vectors.
A self-inactivating MMLV retrovi書秒ral vector is first constructed as a p明開lasmid in E. co見飛li. It is then transfect家了ed into packaging cells along with seve少月ral helper plasmi土月ds. Inside the p拍鐵ackaging cells, vector DNA loca服理ted between the LTRs劇白 is transcribed into RNA, an紙舊d viral proteins志國 expressed by t筆從he helper plasmi化鄉ds further package聽爸 the RNA into virus. Live virus is th上湖en released into th風還e supernatant, which can be u輛下sed to infect target cells directl近開y or after concentrat照媽ion.
When the virus is added to target低靜 cells, the RNA cargo is s開信huttled into cells wher舊兒e it is reverse transcribed into DNA a人雪nd randomly int亮低egrated in the host genome. The non-c學討oding RNA sequence that was place熱都d in-between th兒西e two LTRs during vector construction 男去is permanently insert山醫ed into host DNA alongside the res答身t of viral genome.吧數
By design, self-inactivatin對師g MMLV retroviral vectors lack th著月e genes required for vir電書al packaging and transduc媽街tion (these genes are carried by helpe國女r plasmids or integrated into packagin她議g cells instead).話知 As a result, viruses pro那醫duced from these vectors have the站舞 important safety feature of bei厭木ng replication incompetent (meanin道離g that they can transduce ta生樹rget cells but cannot replicate in the中市m).
For further information about t不地his vector system, please r唱國efer to the papers belo舊家w.
| References | Topic |
|---|---|
| Cell. 157:77 (2通聽014) | Review on non-codi物兵ng RNAs |
| Front Genet. 6:2 (2015) | Review on functionality of non-花短coding RNAs |
| PLoS One. 8:e77070 (2013) | Retrovirus-mediat什上ed expression of l自玩ong non-coding RNA |
| J Virol. 61:1639 (1987)紙月 | Extended packaging signal in報些creases the titer of MMLV vectors |
| Gene Ther. 7:1063 (2000) | Tropism of MMLV vectors depend自司s on packaging cell lines |
| Proc Natl Acad Sci USA. 83:3電體194 (1986) | Designing of self-ina暗黃ctivating retrov什從iral vectors for gene transfer into mam我志malian cells |
亮點
The SIN MMLV retrovirus non-cod不我ing RNA expression vector&nb房術sp;is optimized for high co機務py number replicati麗時on in E. coli, h筆我igh-titer packaging 匠如of live virus, efficient viral transdu銀個ction of a wide range of cells, ef用用ficient vector int司相egration into the host g熱這enome, and high-level expression.
優勢
Permanent integra道說tion of vector DNA: Conventional transfecti農那on results in almost entirel紙新y transient deli長化very of DNA into host cells du姐雨e to the loss of D技水NA over time. This problem is especi雨喝ally prominent in rapidly d到都ividing cells. In 錢明contrast, retrovir遠土al transduction can事站 deliver non-coding R厭金NAs permanently into host 地小cells due to integration of the viral上站 vector into the host genome.
Broad tropism: Our packaging sy森那stem adds the VSV-G envelop pro有玩tein to the viral surface. This p黑愛rotein has broad tropism師身. As a result, cells from窗下 all commonly u吧區sed mammalian species such as human, m大地ouse and rat can be transduced. 靜少Furthermore, many specific 朋姐cell types can be transduced, thoug音吃h our vector has difficulty tran見大sducing non-dividin高河g cells (see disadvantages below).
Customizable internal promoter: 鐘房;Our vector is designed to 事家self-inactivate the promoter activity購西 in its 5' LTR upon integrat件信ion into the genome. As a result, user不影s can put in their own promoter 短術to drive their non-coding RNA with友訊in the vector. This is a distin可船ct advantage over our wildtype照計 MMLV retrovirus vectors, which 內我rely on the promot腦遠er function of 5' 山醫LTR to drive the ubiquit頻事ous expression.
Relative uniformity of deli紅下very: Generally, viral t場自ransduction can deliver vectors i路鄉nto cells in a relative農報ly uniform manner. In contrast, con計生ventional transfecti可對on of plasmid vectors 爸些can be highly non-unif報區orm, with some cell月個s receiving a lot of copies w吧樹hile other cells receivin吧服g few copies or none.
Effectiveness in vitro an頻秒d in vivo: While our vector is mostly used知車 for in vitro tran微錯sduction of cultured cells, it 木微can also be used to transd資從uce cells in live animals.
Safety: The safety of ou筆算r vector is ensured by tw我家o features. One is the partitioning of店西 genes required for麗弟 viral packaging and transduction into能新 several helper plas我船mids; the other is self-inactiva飛信tion of the promoter 知志activity in the長海 5' LTR upon vector integration房水. As a result, it is essenti長了ally impossible for replication co船兒mpetent virus to emerg作校e during packaging and transduction短高. The health risk of working with o光冷ur vector is therefore m報小inimal.
不足之處
Moderate viral titer員西: Viral titer from our vect費從or reaches ~107 TU/ml in the 窗土supernatant of 刀就packaging cells without fur聽醫ther concentration. This is 視錯about an order of ma河懂gnitude lower than our lentiviral vecto身請rs.
More limited cargo space t靜月han wildtype MMLV: The MMLV retroviral gen公音ome is ~8 kb. In our vecto短姐r, the components necess影大ary for viral p科子ackaging and transduction occupy ~2.5麗國 kb, which leaves only ~5.5 kb to acco醫雜mmodate the user'讀匠s DNA of interest, including both the n醫兵on-coding RNA and the p生廠romoter.
Difficulty transducing n舊分on-dividing cells: Our vector has di什村fficulty transducing 公謝non-dividing cells.
Technical complexity: The use of MMLV retroviral vector請朋s requires the production of live vir看湖us in packaging cells followed by the 人看measurement of viral titer.厭身 These procedures are techni舞道cally demanding and time靜煙 consuming relati你黃ve to conventional plas很玩mid transfection.
載體關鍵元件
CMV promoter: Human cytomegalovirus immedi近拿ate early promoter場去. It drives transcription o月物f viral RNA in packaging cel問是ls. This RNA is then packaged 請媽into live virus.
MMLV 5' LTR-ΔU3: A deleted version of the MM錢下LV retrovirus 5' long terminal repea腦嗎t. In wildtype MMLV retrovirus看員, 5' LTR and 3' LTR船請 are essentially identical in se信街quence. They reside on two ends o高如f the viral genome and point in th音市e same direction. Up草知on viral integrati街東on, the 3' LTR sequence 費女is copied onto 煙答the 5' LTR. The LTRs carry both prom個高oter and polyadenylation functi章這on, such that the的妹 5' LTR acts as a promoter to drive 西路the transcription of the viral 嗎是genome, while the 3' LTR acts老短 as a polyadeny公體lation signal to terminate t聽能he upstream transcr黃房ipt. On our vector, MMLV 5' LTR西分-ΔU3 is deleted for a region t煙公hat is required for the LTR都山's promoter activity. This does not 有紅affect the production o街我f viral RNA during packaging becaus書黑e the promoter function is 那銀supplemented by the CMV pro高現moter engineered upstream of Δ自身5' LTR.
Ψ plus pack2: MMLV retrovirus packaging s睡呢ignal required for the p物錢ackaging of viral RNA into viru花下s.
Promoter: The promoter drivi機廠ng expression of your non-coding 男影RNA is placed here.
Non-coding RNA:&n知火bsp;The non-coding RNA of your intere見相st is placed here.
WPRE: Woodchuck hepatitis virus老子 posttranscription森刀al regulatory element. It en刀媽hances viral RNA stability in 視用packaging cells, lea來影ding to higher tit匠很er of packaged virus.
MMLV 3' LTR-ΔU3: A truncated version of長很 the MMLV retrovirus 3' lon樹為g terminal repeat. This leads劇林 to the self-inactivation of 費雪the promoter activity of the微線 5' LTR upon viral小船 vector integration into我雪 the host genome笑店 (due to the fact that 3' LTR is c麗到opied onto 5' LTR during v離放iral integration城東). The polyadenylatio可生n signal contained in MMLV 3' LTR-ΔU妹分3 serves to termin數到ate all upstream transc廠費ripts produced both冷不 during viral packaging 爸從and after viral integration into 事又the host genome.
SV40 late pA: Simian virus 40 late polyadeny店黑lation signal. It further facilita師銀tes transcriptio黑民nal termination after the 3' LTR dur他文ing packaging. This elevates th讀為e level of functional viral RNA音務 in packaging cells, thus improving v玩畫iral titer.
pUC ori: pUC origin of replication. Pl白船asmids carrying 了刀this origin exist in hi了還gh copy numbers友拿 in E. coli.
Ampicillin: Ampicillin resi購線stance gene. It allows the plasmid t可票o be maintained by ampicillin selectio的水n in E. coli.