Regular Plasmid shRNA Knockdow是月n Vector

概述

Our regular plasmi新舊d shRNA knockdown vector s吧知ystem is a simple and e間雪fficient method for transi關黃ently knocking down expression of a t影科arget gene in a wide variet畫多y of cell types. This一麗 system utilizes co短校nventional plasmid transf哥遠ection to introduce an 要筆shRNA expression cass行火ette into mammalian cells for可物 knocking down a訊雪 gene of interest. The s分刀hRNA is expressed from th去光e human U6 promoter, 著市leading to degradation頻到 of the target gene mRNA.

Delivering plasmid vectors into mamm歌火alian cells by conventional transfecti書頻on is one of the笑不 most widely us妹笑ed procedures in bio老坐medical research. Whil明上e several sophisticated ge綠件ne delivery vector systems have been水見 developed over the years such as區器 lentiviral vector和很s, adenovirus ve畫在ctors, AAV vectors and piggyBac, 水大conventional plasmid transfection rem見得ains the workhorse of gene del員北ivery in many labs. This 服什is largely due to it笑學s technical simplici光懂ty as well as good efficiency in a wi爸是de range of cell types. A key featur暗農e of transfecti些從on with regular大拍 plasmid vectors is 草飛that it is transi男微ent, with only a 少短very low fraction of cells 都拍stably integrating the plasmid i去美n the genome (typicall妹西y less than 1%). 村北;

For further inform湖很ation about this vector system, pleas樹朋e refer to the papers below.

References	Topic
Methods Mol Biol. 629:141 (20些門10)	Review on design and delivery of shR輛影NAs
Cancer Gene Ther. 16:807 (2009)	Comparison of siRN少自A and shRNA off target e也到ffects
Mol Cell Biol. 24:2614 (2004)窗黑	Regular plasmid-mediated expres火和sion of shRNAs

亮點

Our vector is optimized分雨 for high copy number re空船plication in E. coli and high-e木市fficiency transfection. Cell問微s transfected with 習計the vector can be selected and/or日妹 visualized based on mar工老ker gene expression as chosen by the海裡 user.

優勢

Technical simplicity: Delivering plasmid vectors 要放into cells by convent懂相ional transfecti短制on is technically straight黑員forward, and far easier than virus-base南的d vectors which require the pac電道kaging of live virus.

High-level expression: Conventional tran我愛sfection of plasmids 學什can often result in 地木very high copy n門林umbers in cells (up to several thousa學醫nd copies per cell). Th媽為is can lead to very high這音 expression levels of th金外e genes carried on the vector.

Ability to add selection 商體markers: Our regular plasmid 地技shRNA knockdown vec很人tor includes the opti厭麗on for users腦愛 to add marker gene樹白s such as a drug selection gene or a v信相isually detectable fluorescence&nbs視妹p;gene. This allo空拿ws cells transfected with the電兵 vector to be selected and/or很離 visualized and therefore provide吧大s a distinct advant討學age over transient knockdown by syn事訊thetic siRNAs where no markers ca懂相n be incorporated.

Reduced off-target錯照 effects: shRNAs have been件水 seen to have reduced off-t西了arget effects compared to synthetic si些月RNAs since a much hig謝開her concentration of siRNA is requir朋計ed to achieve comparable 吧生levels of knockdown obtaine遠暗d with an 飛有shRNA construct, therefore potentia紙知lly leading to increased of說一f-target effects. Addition了秒ally, unlike shRNAs which are transcr錯船ibed in the nucleus, siR在明NAs remain in the cytopl不報asm and are suscepti到街ble to degradation which could ha討他ve further undesira公爸ble targeting effects.

不足之處

Non-integration of vector DNA: Conventional transfection of plas自動mid vectors is also 家新referred to as transient tra拍電nsfection because the vector stays mos房藍tly as episomal D場慢NA in cells without integration. 資大As a result, the regular plasmid shR雨議NA vector can k放從nock down genes of in員喝terest transiently in mammalian c睡綠ells. However, pla間學smid DNA can integrate perma城刀nently into the 信如host genome at a very l店民ow frequency (one玩站 per 10² to 10⁶ cells depending on 好月cell type). If a drug resistance or f司短luorescence marker is incorporated int笑船o the plasmid, cells stably integr冷頻ating the plasmid can be derived b議睡y drug selection or cell sorting af要錢ter extended culture.

Limited cell ty就家pe range: The efficiency of plasmid t嗎務ransfection can vary greatly f大林rom cell type to cell type. Non-divi商呢ding cells are often mor兵但e difficult to transfect than divi照還ding cells, and primary cells are of黃吧ten harder to transfect than imm要森ortalized cell lines. Some importan得章t cell types, such as ne個你urons and pancreatic β cells, are noto劇紙riously difficult to tr業喝ansfect. Additionally, plasmid transf冷身ection is largely下知 limited to in vitro applicatio雜上ns and rarely use志兒d in vivo.

Non-uniformity of gene deliv和山ery: Although a successful transfec資窗tion can result in very high女計 average copy number of the tra學舞nsfected plasmid 遠窗vector per cell, this can be highl器這y non-uniform. Some cells can carry訊刀 many copies while others c身購arry very few or none. This城長 is unlike trans自湖duction by virus-based vectors which我厭 tends to result in r紅美elatively unifor信算m gene delivery into現票 cells.

載體關鍵元件

U6 Promoter: Drives expression of the shRNA. This is船林 the promoter of t看見he human U6 snRNA gene, an RNA polymera愛長se III promoter which efficien為子tly expresses short RNAs.

Sense, Antisense: These sequences a東農re derived from your targe不分t sequences and are transcribed t遠店o form the stem portion 森化of the “hairpin” stru喝空cture of the shRNA.

Loop: This optimized sequence姐東 is transcribed to form the作秒 loop portion of the shRNA “hairpin” 商知structure.

Terminator: Terminates transc看你ription of the shRNA.

hPGK promoter: Human phosphoglycerate kinase 1 ge習事ne promoter. It drives the ubiquit做吃ous expression of the downstream marke爸道r gene.

Kozak: Kozak consensus sequence. I答人t is placed in front 商銀of the start codon 家時of the ORF of interest because it is b得雜elieved to facilitate t媽知ranslation initiation in eukaryotes.

Marker: A drug selection gene (such as 做我neomycin resistance) or a visually de得年tectable gene (such as EGF們問P). This allows cel拍金ls transfected with the vector to電空 be selected and/or visualized.

SV40 late pA: Simian virus 呢靜40 late polyadenylation signal懂不. It facilitates tran歌要scriptional terminatio刀也n of the upstre下廠am ORF.

pUC ori: pUC origin of re得放plication. Plasmids ca電朋rrying this origin exist in high copy 紅一numbers in E. coli.

Ampicillin: Ampicillin resi就在stance gene. It allow醫子s the plasmid to be main理外tained by ampicillin se房志lection in E. coli.

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Regular Plasmid shRNA Knockdow是月n Vector