MMLV retrovirus Non-Cod師現ing RNA Expression Ve能老ctor

The MMLV retrovirus non-coding數老 RNA expression vector 朋媽is a highly efficient v商票ehicle for permanently introducing non錢河-coding RNAs of interes音校t in mammalian 雨報cells. Non-coding RNAs include a 大我wide variety of short (<30 nucle化廠otides) and long (農校>200 nucleotides) funct舞訊ional RNA molecules such as mi業和cro RNAs (miRNAs)吧裡, small interfering RNAs (siRNAs), p窗去iwi-interacting RNAs (piR動雜NAs), small nuclear RNAs (snRNAs輛內), small nucleolar RNAs (snoRNAs), larg新下e intergenic non-co校信ding RNAs (lincRNAs), intro對路nic long non-co放看ding RNAs (intronic lncRNAs), 笑爸natural antisense trans電厭cripts (NATs), enhanc睡鐵er RNAs (eRNAs) and promoter-associated玩一 RNAs (PARs), none of which ar業學e translated into proteins, h事這owever have been fo一煙und to play important roles新通 in many cellular processes such as DNA鐵志 replication, epigenetic regulation, tr音他anscriptional and p在學ost-transcriptional regulation and tra舞河nslation regulation.

The MMLV retrovi白船rus non-coding RNA expres物謝sion vector uses the ubiquitous promote錯月r function in the 5' LTR&nbs著作p;of the MMLV retroviral genome to dri腦那ve the expression of the user-select計你ed non-coding RNA 作化gene, which is mediated 離友by RNA polymerase II-d小白ependent transcription. For RNA pol些筆ymerase II-mediated transcripti刀錢on, the start site體站 is typically in the黑從 3' region of the prom鄉時oter while the termination site is wi喝吧thin the polyA signal sequence. As a re校兒sult, the transcript generated f西腦rom this vector does not co雜很rrespond precisely to the select機如ed non-coding RNA gene, but contain用鐘s some additional se學紅quences both upst弟煙ream and downstream. 

An MMLV retroviral vector is first謝去 constructed as a plasmid in E廠媽. coli. The non-coding RNA of intere風是st is cloned between the t對銀wo long terminal repea做煙ts (LTRs) during 不南vector construction放話. It is then transfected 空見into packaging cells alon看到g with several he制靜lper plasmids. Inside the packagin低多g cells, vector DNA locat如靜ed between the LTRs i可美s transcribed into RNA, and viral prot下從eins expressed by the he線理lper plasmids further package th高鐘e RNA into virus照光. Live virus is then released 計樹into the supernatant, which can be u少離sed to infect target cells direct相樹ly or after concentration.

When the virus is added to target 光畫cells, the RNA cargo is shuttled不鐵 into cells where it is reverse trans來秒cribed into DNA and randomly integr視草ated in the host geno藍工me. The non-cod森雨ing RNA sequence that was placed房店 in-between the 報的two LTRs during ve些那ctor construction is permane子長ntly inserted into host DNA 日校alongside the rest of 女訊viral genome.

By design, MMLV retroviral 空熱vectors lack the genes required for v錢美iral packaging and t人見ransduction (these genes a和遠re carried by helper pl新姐asmids or integrated into p民個ackaging cells instead). As a re歌多sult, viruses produced from 購哥the vectors have the imp冷拍ortant safety feature 車光of being replication incompetent (mean也看ing that they can transd得男uce target cells but c車器annot replicate in them).

For further information about thi你南s vector system, please refer to 還頻the papers below.

Permanent integration of ve關低ctor DNA: Conventional t報湖ransfection results in不通 almost entirely transient delivery 村長of DNA into hos讀月t cells due to the loss of DNA over t近船ime. This problem is es路國pecially prominent i報好n rapidly dividing ce習小lls. In contrast, r愛山etroviral transduction can deliver用又 genes permanently into h紙化ost cells due to integration 和慢of the viral vector into the host 醫店genome.

Broad tropism: Our packaging system a師樹dds the VSV-G envelop protein t頻習o the viral surfa多道ce. This protein has broad tropism.紅麗 As a result, cells from all commonly西船 used mammalian species such as h們銀uman, mouse and rat can be transduced. 地友Furthermore, almost any mammalian c兵科ell types can be transduced, thoug生睡h our vector has difficulty transduci森路ng non-dividing cells (se到錢e disadvantages below).

Large cargo space: The wildtype MMLV retroviral genome i道嗎s ~8 kb. In our vector, the compo公通nents necessary for viral packaging an下通d transduction occupy ~2.5 kb, w呢放hich leaves ~5.5 kb to 分工accommodate the user'作但s DNA of interest. Because our vector校拿 is designed for the inse花電rtion of only a 刀鄉non-coding RNA sequence, 書森this cargo space is sufficient for 一下most applications.

High-level expr東白ession: The 5' LTR contains來學 a strong ubiquitous promoter計從 that drives high-leve林報l expression of t科跳he user's non-coding RNA 暗嗎of interest.

Relative uniformity of gene delivery: Generally, viral tran行見sduction can deliver vectors int低章o cells in a relatively uni的地form manner. In contrast, convention書來al transfection of plasmid v工從ectors can be highly 近花non-uniform, with some資錯 cells receiving a lot of copies 哥路while other cells receiving村友 few copies or none.

Effectiveness in爸多 vitro and in vivo: While our vector is mostly 短妹used for in vitro transduction o司計f cultured cells, it c美件an also be used to transduce cel服這ls in live animals.

Safety: The safety of our v睡會ector is ensure關通d by partitioning gen農鐘es required for viral packaging an少影d transduction into廠不 several helper plasmids or inte多道grating them in好東to packaging cells. As a result,問務 live virus produced from our vector is草日 replication incompetent.

Dependence on 5' LTR村河 promoter: Expression of the non-c拿個oding RNA of inter中習est in our vector is driven 是聽by the ubiquitous promoter function好她 in the 5' LTR. Thi秒事s is a distinct disadvantage 報低as compared to our lentiviral vectors資西 which allow the 話站user to put in their own p知分romoter to drive their gene of inter那雨est.

Moderate viral titer: Viral titer from our vector rea綠快ch ~10⁸ TU/ml in the supernatan高見t of packaging cells withou森離t further concentration. This is abou木船t an order of magnitude lower than票知 our lentiviral vec身術tors.

Difficulty transducing non-divid書能ing cells: Our vector has difficulty 件吧transducing non-dividing 技懂cells.

Technical complexity: The use of MMLV retroviral vect街金ors requires the production of live v刀上irus in packaging cells f雜舞ollowed by the measurement o多秒f viral titer. These procedures are理妹 technically demanding時放 and time consuming re湖腦lative to conventional plasmid trans城相fection.