PiggyBac Tet Regulatory Protein Expr西文ession Vector

VectorBuilder’s p說道iggyBac Tet regula購拿tory protein ex報喝pression vector can be used for trans森樂fection-based, perma哥紅nent delivery of 子會Tet regulatory protein(s) (tTS,醫林 rtTA, etc.) into m們視ammalian cells to help you achiev行師e tetracycline-inducible expression小說 of target genes place多店d downstream of a tetracycline朋媽 responsive-element (TRE)山金 promoter.

The Tet-On inducible system is a power紙花ful tool to control the timing o子相f expression of the gene(s) of inter是答est (GOI) in mammalian cel懂慢ls. Our Tet-On inducible gene 通資expression vectors are designed to ac風媽hieve nearly complete si藍議lencing of a GOI in 公請the absence of tetracycline an麗很d its analogs (e.g. doxycyclin是新e), and strong, rapid expressio看紙n in response to the addition of tetrac鄉民ycline or one of its analo分數gs (e.g. doxycycline). This 水子is achieved through a multicomponent 和年system that incorporates active si西冷lencing by the tTS protein in綠和 the absence of tetracycline and stro做著ng activation by the rtTA pr會外otein in the presence 又女of tetracycline. In the ab舞做sence of tetracycline, the tTS pr風物otein derived from the fusi可站on of TetR (Tet repressor p請暗rotein) and KRAB-AB 東外(the transcriptio數中nal repressor domain of Kid-1 protein) 對樹binds to the TRE p自風romoter, leading to the ac工看tive suppression of gene transcription.農秒 The rtTA protein, on謝畫 the other hand, derived from the fu這月sion of a mutan雪吃t Tet repressor and 聽科VP16 (the transcription act站影ivator domain of virion protein 1間業6 of herpes simplex virus), binds 區師to the TRE promoter 水微to activate gene 數西transcription only 鐵資in the presence of tetracyclin那厭e.

While our piggyBac Te舞聽t regulatory protein ex服謝pression vector i雪雨ncludes an expression cassette co路湖nsisting of the Tet regulatory車計 protein(s) driven by a user-sel友呢ected promoter, the 中也GOI driven by t懂朋he TRE promoter must be provided 很東using a separate helper vector to a低答chieve tetracycline-induced ge哥你ne expression in the presence of tetra高的cycline, while minimizing 樹書leaky expression in the absen術相ce of tetracycline藍化.

Our piggyBac inducible gene expr聽秒ession system comprises two c村醫omponents: the transpo影熱son plasmid and the t少對ransposase (helper PBase). Th站水e transposon plasmid 鐵些contains two term國文inal repeats (TRs) bracketing th也分e region to be trans購一posed. The Tet regulatory prot會城ein expression cassette is cloned in為黑 between the two TRs. T鐘裡he transposase can be 通暗delivered into target cells明是 through two methods. The he妹那lper plasmid can be transientl海玩y transfected into ce站錯lls. Alternatively, target cells can b男廠e injected with tr自業ansposase mRNA generated by in v師也itro transcription from the helper plas間去mid. When the helper PBase如音 and the piggyBac習紙 transposon vector are co-introdu草作ced into target cel科文ls, the transposase produced from the要看 helper would recogniz來子e the two TRs on the transposon and 書開insert the flan村關ked region including the two TRs i線自nto the host genome. Inse藍錯rtion typically occurs at host chrom那多osomal sites that contain the TTAA seq這銀uence, which is duplicat妹美ed on the two flanks門用 of the integrated fragment. Through 還弟both methods of delivering trans務電posase, it is expressed for山關 only a short time議數. Upon the loss of the helper plas拍南mid or degradation of t睡作ransposase mRNA司市, the integration是銀 of the transposon into t報遠he host genome becomes perm唱站anent.

PiggyBac is a class II transpos房民on, meaning that 雪刀it moves in a cut報也-and-paste manner, hopping from place相要 to place without leaving copies 師木behind. (In contra空舊st, class I tra相們nsposons move in a 相還copy-and-paste manner.) If the tran日好sposase is reintroduc文工ed into the cel拍火ls, the transposon could get e腦民xcised from the genome數說 of some cells, footprint-舞中free.

For further information 煙唱about this vector system, plea還從se refer to the papers below.