PiggyBac miR30-B放知ased shRNA Knockdown Vector

The piggyBac miR30-based shR關資NA knockdown vec員到tor system is a simple and eff水樂icient method for stably&nb術照sp;knocking down又校 expression of target gene(s) i現人n mammalian cells. This transpos行喝on-based system util訊媽izes plasmid transfection (rather than 討下viral transduction) to permanent冷答ly integrate a polyc影的istronic expressi慢飛on cassette consisting of one or m是刀ore miR30-based shRNAs (藍木shRNAmiR) targeting gene(s) of inter還內est and a user-熱店selected ORF into the hos黑都t cell genome. The s時冷hRNAmiR transcript is processed她空 by endogenous, cellular mi又高cro-RNA pathways to produce mat車喝ure shRNAs, which facilitate de鐵習gradation of target北理 gene mRNAs. The pe得弟rmanent nature of knockdown鐵裡 by the piggyBac syst術如em has several m場得ajor advantages over tr通動ansient knockdow高海n by synthetic siRNA (票物see Advantages secti鐵我on below).

The piggyBac miR30-based shRNA k姐還nockdown vector syst你我em contains two vectors, both engin頻體eered as E. col讀報i plasmids. One vector, referred to a在作s the helper plasmid, enc不相odes the transposase. The厭答 other vector, referred件到 to as the transposon plasmid,那內 contains two terminal repeats (TRs) 慢樹bracketing the regi藍開on to be transposed, whic理村h includes the polycis業城tronic shRNAmiR and ORF expression 友朋cassette. W為坐hen the helper an刀家d transposon plasmids 制玩are co-transfec電雨ted into target cel森爸ls, the transposase produced from th了筆e helper plasmid recognizes the tw廠大o TRs on the transposon, and inserts t動我he flanked region inclu男明ding the two TRs 村放into the host genome.來志 Insertion typically 為如occurs at host chromosomal sites that美藍 contain the TTAA sequence, which 藍拿is duplicated on雪很 the two flanks of the integ現行rated fragment.

Unlike conventional shRNA vect林兵ors, which utili煙媽ze RNA polymerase人國 III promoters such as U6, miRNA-日行based shRNA systems影拍 are placed under the control of 玩著standard RNA polymerase II promot一樂ers. This allows the use of tissue-spec這什ific, inducible, or variable-strength p事日romoters, enabling a variety of exper計們imental applications not possible w外數ith constitutive U6討玩 promoters.

The ability of RNA polymeras地從e II promoters to 暗音efficiently transcribe long transc些開ripts in the miRNA-based 少看shRNA systems also provid年內es additional advantages re中術lative to other knockdown vecto拿樂r systems. Multi風醫ple shRNAmiRs can be紅我 transcribed as a single polycistron,一短 which is proce飛新ssed to form mature shRNA畫學s within the cell. This allows knoc白老kdown of multiple genes or targeting為些 of multiple regions wi廠資thin the same gene us文這ing a single transcript個體. As a result, this vector is avail街購able for expressing either singl那雜e or multiple shRNAmiRs. Secondly,唱外 in this vector system, a user-select到著ed protein coding gene is also pos時麗itioned within the same p爸海olycistron as the shRN技著AmiRs. The expression of this ORF哥舞 can be used to directly 畫動monitor shRNA tra城你nscription (if a ma路視rker ORF is used) or can be 現訊used for other purposes r很線equiring co-expre子中ssion of an ORF and shR兵時NA(s). 

PiggyBac is a class II transp樂年oson, meaning that it moves in a cu玩她t-and-paste mann又我er, hopping from電慢 place to place withou歌西t leaving copies behind. (In con坐風trast, class I transposons move 書話in a copy-and-paste m要日anner.) Because微嗎 the helper plasmid 件子is only transiently trans房低fected into host cell業微s, it will get lost over time. 有小With the loss of the helper plasmid, 木我the integration of the業跳 transposon in the genome of host ce去請lls becomes permanent. If these cell術資s are transfected with the 玩子helper plasmid again, the睡頻 transposon coul分坐d get excised from the genome of s對頻ome cells, footprint金志 free.

For further inf近兵ormation about 但快this vector system, please refer to讀女 the papers below.

Promoter choice: Unlike standard shRNA systems, w水鐘hich utilize RNA 師好polymerase III promoters影樹 such as U6, miR30-based喝高 shRNAs can be transcribed by diverse R少問NA polymerase II promoters. This also e場資nables the use of tissue-specific有明 or inducible promoter身會s.

Multiple shRNA co-expre男市ssion: Because RNA polymerase II ef窗呢ficiently transcribes long RN大樹As, multiple shRNA要來miRs can be expressed as a坐什 polycistron from a si讀輛ngle promoter. 照輛Therefore, this v那愛ector is available for e南煙xpressing either single or multiple shR西人NAmiRs.  

Co-expression of a repo窗子rter ORF: A user-selected錢風 gene of interest or repor微習ter gene ORF is 紙音co-expressed with the shRNAmiRs, as 視綠a polycistron. This facilitates d很小irect monitoring of shRNA 房技transcription.

Permanent integration and knockdow生店n: Conventional票說 transfection results in almos家從t entirely transien飛間t delivery of DNA into hos海視t cells due to the loss of DNA over 志員time. This problem is especially 對拿prominent in rap師厭idly dividing c城日ells. In contrast, tr資筆ansfection of mamm姐房alian cells with the地訊 piggyBac transposon plasmid alon金子g with the helper plasmid can deliver 開少DNA sequences carr場中ied on the transposon permanent遠愛ly into host cells due t水如o the integration of the transpos吃微on into the host genom討拿e. As a result, the knockdow議明n of the target gene(s) achieved with市金 this vector is stable and permanent. 知訊This can be an important advantage f下電or many experimenta議商l goals. It allo風線ws long-term anal唱去ysis of the knock視都down phenotype. It facili店會tates the isolation of clones森湖 having different levels of 唱了knockdown and/or different phen鄉們otypes. When the knockdown 鐘玩vector carries a fluorescence mar學但ker such as EGFP, it also allows cel兒弟ls with different 些小amounts of tran匠議sposon integrati信視on (and hence po去公tentially different levels of k明那nockdown) to be isolated by flow sorti上金ng cells with different f跳土luorescence intensity.妹村

Reversibility: If cells carrying a pi購唱ggyBac shRNA transposon 公們are transfected with 妹報the helper plasmid ag家拿ain, the transposon may be e和樂xcised from the genome of s購物ome cells, footprint free, el哥和iminating expression of the shRNA fr門購om those cells. However, this will個問 occur in only a subset o慢老f cells.

Technical simplicity: Delivering plasm一有id vectors into cells b有高y conventional transfection i亮行s technically straigh計物tforward, and far easier than virus女多-based vectors which require the pack都個aging of live virus.

Safety: Conventional transfection does not hav議醫e the safety concerns which are 我行often associated with viral v暗玩ectors.

Limited cell type range:區自 The delivery o區就f piggyBac vectors i如報nto cells relies on transfectio資科n. The efficiency of transf行鐵ection can vary greatly from cell type數場 to cell type. Non-divi了海ding cells are of服紅ten more difficult做日 to transfect tha船小n dividing cells, and primary體時 cells are often hard她分er to transfect than immortalized c舞人ell lines. Some impor科少tant cell types, such as neu商錯rons and pancreatic β cells近妹, are notoriously difficult to transfec下玩t. Additionally,窗下 plasmid transfection跳開 is largely limited to照去 in vitro applicatio音能ns and rarely used in vivo. These 在黑issues limit the use 西弟of the piggyBac system.