Tol2 miR30-Based shRNA Knockd弟不own Vector

The Tol2 miR30-based sh裡影RNA knockdown vector system is媽少 a simple and efficient method for stab資員ly knocking down expression of tar林森get gene(s) in mammalian cells. T音小his transposon-based system utilizes p得事lasmid transfection (rather吃讀 than viral transduction資爸) to permanently integra姐線te a polycistronic妹視 expression cassette consistin為購g of one or more miR30-based sh車喝RNAs (shRNAmiR) targeting gene(s)說著 of interest and a us冷自er-selected ORF into the ho文我st cell genome. The shRNAmi月請R transcript is processed 從機by endogenous, cellular mic女信ro-RNA pathways to produc吧綠e mature shRNAs, w舞得hich facilitate deg兒拿radation of target gene mRNAs.&nb少跳sp;The permanent nature of kno這新ckdown by the Tol2 system 相理has several major adva物事ntages over transient knockdown by 弟關synthetic siRNA (see Advantages s黃理ection below).

The Tol2 miR30-based shRNA 快件knockdown vector 分藍system contains two v廠小ectors, both engineered as E. 在學coli plasmids. On海秒e vector, referred to as the helpe低場r plasmid, encod資我es the transpos湖影ase. The other vecto下房r, referred to as the transpo影工son plasmid, contains tw媽司o terminal repeats (TRs)一到 bracketing the 通南region to be tr個習ansposed, which inclu冷那des the polycistro議來nic shRNAmiR and ORF e友道xpression cassette. When the 就綠helper and transposon plasmi家得ds are co-transfected into t道樂arget cells, the transposase produ話短ced from the helper plasm我嗎id recognizes the two TRs on the tra花可nsposon, and inserts the flank機藍ed region including the tw制我o TRs into the 還一host genome. Insertion oc雜員curs without any sig問坐nificant bias with respect to inserti冷大on site sequence. This is unlike t懂年ransposon syste妹商ms which have specific target consens文在us sites. For example, p對樹iggyBac transposons typically inserts 通人at sites containing the sequence TTAA.

Unlike conventional shRNA vector區體s, which utilize RNA po藍討lymerase III promoters such村好 as U6, miRNA-based shRNA systems are 分那placed under the control of 女對standard RNA polymerase II p件劇romoters. This al店厭lows the use of tissue-specifi訊鄉c, inducible, or variable-str工雪ength promoters, enabling a va從風riety of experimental applicati風技ons not possible wit林冷h constitutive U6 promoters.

The ability of RNA po算你lymerase II promoters t綠為o efficiently transcribe long 冷如transcripts in the miRNA-based s開路hRNA systems also prov間水ides additional adv路對antages relative 微遠to other knockdown vector systems.飛一 Multiple shRNAmiRs can be transcrib愛唱ed as a single polyci坐費stron, which is proces但文sed to form mature shRNAs within the c去說ell. This allows knockdown of 妹空multiple genes or targe林開ting of multiple re在媽gions within the 科議same gene using a singl空醫e transcript. As a result年司, this vector is available 內拿for expressing either s農綠ingle or multiple shRN視離AmiRs. Secondly, in this vect上紅or system, a user-sele時問cted protein co離子ding gene is also po河化sitioned within the same p快河olycistron as the shRNAmiRs. 高身The expression of this ORF can be u錯森sed to directly monitor shRNA t錯頻ranscription (if a marker ORF is used)器車 or can be used for other purposes 聽長requiring co-exp中小ression of an OR裡火F and shRNA(s). 

Tol2 is a class II transposon, mea問的ning that it moves in a鐘友 cut-and-paste mann紅快er, hopping from電國 place to place without leaving c件雪opies behind. (In contrast,頻信 class I transposons move in a copy-a內聽nd-paste manner.) Tol2 窗他integrates as a sin跳草gle copy through a 話民cut-and-paste mechanism. 中紙At each insertion site, the Tol2 t坐議ransposase creates an 8 bp duplication冷師, resulting in identical 8 bp河化 direct repeats flankin山在g each transposo路森n integration site in the genome.

For further information abou光弟t this vector system, please re下慢fer to the papers below.