AAV miR30-Based 人子shRNA Knockdown 店制Vector

Our AAV miR30-based shRNA kno很話ckdown vector system 工爸is a highly efficien友年t viral tool for knockin長人g down expression of 懂就target gene(s) 和短in vitro or in vivo. Due to the low i些中mmunogenicity and cytotoxicity新綠 of AAV, this is the ideal shRNA vect個地or for many ani錯計mal studies.&nb開內sp;It utilizes AAV-mediated d請冷elivery of a pol拍制ycistronic expr煙不ession cassette consisting of one or內身 more miR30-based sh你些RNAs (shRNAmiR) targeting gene(s) 做她of interest and煙鐵 a user-selected ORF, 一綠where the vector re少愛mains as episomal現校 DNA without integration into the 書票host genome. The shRNAmiR transcr黃請ipt is processed筆跳 by endogenous, cellular micro-RNA 電鐘pathways to prod還話uce mature shRNAs, 計說which facilitate degradation of 物錯target gene mRNAs. 暗你

An AAV miR30-based 我跳shRNA knockdown地長 vector is first答城 constructed as a plasmid in E. co雨議li. The polycistronic expression 外關cassette consisting of one or more紅鐘 shRNAmiRs targ習跳eting gene(s) of inte舞身rest and a user-selected ORF is clon愛黃ed between the two inverted ter熱吧minal repeats (ITRs).&山是nbsp;It is then transfected into packag好女ing cells along wi子遠th helper plasmids, 國廠where the region 她匠of the vector between the two西熱 inverted terminal repeats (吃明ITRs) is packaged into live virus.&nb生購sp;The shRNAmiR exp船坐ression cassette placed in-between th笑得e two ITRs is introduced時拿 into target cells along with the rest南黃 of viral genome.

The wild-type AAV genome is a linear s門司ingle-stranded DNA (ssDNA) wit話車h two ITRs forming a hairpin struc年樹ture on each end. I城湖t is therefore also k關聽nown as ssAAV. In 腦呢order to express genes o子離n ssAAV vectors in host cells, the 放朋ssDNA genome needs t兒小o first be converted to double-stran森農ded DNA (dsDNA) th放草rough two pathways: 1) synthesis o玩到f second-strand DNA 她風by the DNA polymerase machinery of hos冷算t cells using the existing ssDNA ge光章nome as the templat窗影e and the 3' ITR 務子as the priming site; 2) fo月技rmation of intermole厭黃cular dsDNA between the plus- and mi日低nus-strand ssAAV genomes. The for吃照mer pathway is the dominant one.

AAV genomic DNA影國 forms episomal conca拿商temers in the host cell nucleus. In n信家on-dividing cells, these c票微oncatemers can remain for the life of t這身he host cells. 但雜In dividing cells, AAV DNA is lost雪票 through the dilution effect of黑通 cell division, because the e用笑pisomal DNA does not rep刀窗licate alongside ho近我st cell DNA. Random integration of AAV黑場 DNA into the host genome女看 can occur but is extremely 是短;rare. This is desirable in many ge劇務ne therapy settings where the potentia謝靜l oncogenic effect of vecto什筆r integration can pose a signif雪刀icant concern.

Unlike conventional shRN上腦A vectors, which utilize RNA polymera上校se III promoters such as U6土好, miRNA-based shRNA s高自ystems are plac水請ed under the control of stand拍年ard RNA polymera放快se II promoters. This allows the use有志 of tissue-specific, inducible,船路 or variable-strength promoters, enab門廠ling a variety of experiment呢視al applications not possible事麗 with constitutive U6 promoters.

The ability of RNA polymerase務那 II promoters to efficiently transc亮這ribe long transcripts in票從 the miRNA-based sh司這RNA systems also provides addition門校al advantages rel雜兒ative to other knockdown vec聽票tor systems. Multipl女近e shRNAmiRs can be transcribed還哥 as a single polycistron, which i說件s processed to form mature 北店shRNAs within the cell. This al的西lows knockdown of multiple ge爸志nes or targeting of multiple regio河來ns within the same gene using a s還務ingle transcript. As a result, this喝器 vector is available fo商外r expressing either single or mu多通ltiple shRNAmiRs. Secondly,術工 in this vector system, a user-selec校分ted protein coding ge近銀ne is also positioned機術 within the same po音靜lycistron as the shRNAmiRs. The express民員ion of this ORF can be used to dir紅路ectly monitor shRNA transcription (i木會f a marker ORF is used) o費自r can be used for other少窗 purposes requiring co-e歌業xpression of an ORF and shRNA也嗎(s).

A major practical advantage of AA高務V is that in most cases AAV 遠呢can be handled in biosafet章刀y level 1 (BSL1) facilities. T科高his is due to AAV being 的信inherently replication-deficient, pro有黑ducing little or no inflammatio對雜n, and causing no known hu上高man disease.

Many strains of AAV h校電ave been identified in nature. They a小可re divided into different serotypes bas又門ed on different antigenicity of th體林e capsid protein on年師 the viral surf劇民ace. Different sero山亮types can rende紙現r the virus with different tissue tr中動opism (i.e. tissue specific黃師ity of infection). Whe人票n our AAV vectors are packaged房聽 into virus, different serotyp學會es can be confe就會rred to the virus by using different又地 capsid proteins for th錯外e packaging. During cl服女oning, ITRs from AAV2 are used, as thi相森s is common practice in問要 the field and does not impact 資學specificity. Packaging helper plas木森mids include a Rep男得/Cap plasmid, con理舊taining the repli器農cation genes from AAV2 and the caps雪國id proteins for a chosen serotype to雪唱 determine tropism. The們銀 table below lists different 謝但AAV serotypes and their tissue tropis市相m.

For further information about t購行his vector system,長鐘 please refer to the遠雪 papers below.

Our AAV miR30-based shR一影NA knockdown v說我ector incorporate件車s an optimized micro-RNA sys聽少tem for knockdown of target gene(s舊土) and is optimized for high copy number西森 replication in E. coli, high-tit麗美er packaging of live vir坐空us, efficient transduction資可 of host cells, and high-l民外evel transgene expression. 從放This viral vector can be packaged i習媽nto virus using all known capsid serot雪不ypes, is capable of very high件文 transduction efficienc舞通y, and presents low sa通工fety risk. A user-sele可件cted promoter drive藍筆s expression of a poly吧醫cistronic expression casset黑跳te containing a user-selected 舊煙ORF and one or more shRNAmiRs w資市ith optimized miR30-based seq車人uences to mediate efficient shRNA坐長 processing and target ge訊但ne(s) knockdown.

Promoter choice: Unlike standard s也好hRNA systems, which utilize RNA pol場刀ymerase III promoters such as 樹水U6, miR30-based shRNAs can be transc秒舞ribed by diverse RNA poly拍錢merase II promoters. This also 車鐘enables the use o家線f tissue-specific or inducible promote喝術rs.

Multiple shRNA co-expression: 空藍;Because RNA polymerase II長鐘 efficiently transcribes long RNAs, mul時新tiple shRNAmiRs can be expres綠服sed as a polycistron from a sing為照le promoter. Therefore, this vec中花tor is available for expr行睡essing either single or mult舊關iple shRNAmiRs. &nbs低行p;

Co-expression of a reporter 日森ORF: A user-selected gene謝跳 of interest or快道 reporter gene ORF is co-expressed腦土 with the shRNAm玩讀iRs, as a polycist業書ron. This facilitates direct monitor科們ing of shRNA transcription.

Safety: AAV is the safest viral vector system校朋 available. AAV 不不is inherently replic笑東ation-deficient and is no費公t known to cause an一術y human diseases.

Low risk of host genome disruption:&nbs知月p;Upon transduction into host cells, AAV車刀 vectors remain as ep聽哥isomal DNA in the nucleus. The 在美lack of integration int很中o the host genome can be a d生拿esirable feature for 河務in vivo human ap近家plications, as it reduces the開姐 risk of host genome那姐 disruption that might lead to cancer.

High viral titer: Our AAV vector can be packaged低麗 into high titer virus. W好雨hen AAV virus is obtained through our v討學irus packaging service, titer can 學說reach >10¹³ genome copy per ml (GC道公/ml).

Broad tropism: A wide range of cell and tissue站光 types from commonly used mammali做票an species such as human, 術說mouse and rat can be 志歌readily transduced街土 with our AAV vector when it is 農訊packaged into the appropriate 購外serotype. But some cell type兵理s may be difficult to tran地睡sduce, depending on the sero窗雨type used (see disadvantages below年舞).

Effectiveness in vitro 很雜and in vivo: 麗爸Our vector is often us還姐ed to transduce 議報cells in live a件店nimals, but it can also be used effecti章很vely in vitro.

Single miR30-shR醫她NA AAV shRNA knockdown v裡土ector

5' ITR: 5' inverted terminal repeat.為爸 In wild type virus, 5' I一友TR and 3' ITR are e人友ssentially identical in sequence. Th我議ey reside on two ends近話 of the viral genome 商匠pointing in opposite di報飛rections, where they serve as the or懂服igin of viral genome replication.

Promoter: Drives transcription of th議煙e downstream ORF and shRNAmiR polycis道錯tron. This is an RNA polymer林自ase II promoter, rather than an R月木NA polymerase III promoter su睡筆ch as U6.

Kozak: Kozak consensus 校鄉sequence. It is placed in fron熱錢t of the start 筆錯codon of the ORF of interest because i從村t is believed to facilitate translati如笑on initiation in eukaryotes.土山

ORF: The open reading fram現吧e of your gene of inter冷化est or reporter gene is placed唱笑 here. This can be used to monitor sh雜愛RNA expression.腦多

5' miR-30E: An optimized version of the hu街務man miR30 5’ context s黃議equence. Facilitates maturation and p報草rocessing of the shRNA and sep就又aration from the tandem道車ly transcribed ORF and船從 other shRNAs.

3' miR-30E: An optimized version o店開f the human miR30 3’ context sequence個腦. Facilitates maturation and proces雜對sing of the shRNA and separation from 錯頻the tandemly tran短老scribed ORF and other shRNAs.

miR30-shRNA:&nb員視sp;This sequence is deriv作生ed from your target sequence and i在美s transcribed to form the ste討車m portion of the “hairpin” structu章月re of the shRNA.

Regulatory elem為和ent: Allows the user to add 們現the Woodchuck hep對也atitis virus po話笑sttranscription秒討al regulatory e玩短lement (WPRE). WPRE enhances 廠民virus stability in pac男件kaging cells, leading to higher tit文得er of packaged virus and enh業這ances expression of transgenes.

BGH pA: Bovine gr照冷owth hormone polyadenylation signal.弟花 It facilitates transcription一兒al termination and polyade電師nylation of the upstr地秒eam ORF and shRNA愛但miR polycistron.

3' ITR: 3' inverted terminal 朋秒repeat. See description for 5’ ITR.

Ampicillin: Ampicillin resista南微nce gene. It allows the光會 plasmid to be ma銀低intained by ampici場吃llin selection in E. coli.學林

pUC ori: pUC origin of repli音紙cation. Plasmids carrying音人 this origin exist in high copy number和風s in E. coli.