PiggyBac Inducible Gene Expressio子什n Vector (Tet-On, Low Leak)

The PiggyBac ind媽森ucible gene expression vector co年媽mbines VectorBuilder’s highly effic自藍ient PiggyBac vector system and the 鄉子Tet-On inducible gene expressio會錢n system to help yo離著u achieve transfection-base體現d permanent integ答討ration of tetracycline-inducible一木 gene expression cassettes 少術into the host genome.

The Tet-On inducible gene ex開城pression system is a powerf輛現ul tool to control the 務要timing of expression of the gene(s) of工坐 interest in mammalian c志志ells. Our Tet-On induc通農ible gene expression vectors ar雪兵e designed to achieve nearly complete亮外 silencing of the gene(s) of interest 就雨in the absence 水制of tetracycline and it就文s analogs (e.g. doxycycl金商ine), and strong, rapid expressi些弟on in response to the addition of tetr習飛acycline or one of its 市器analogs (e.g. dox們文ycycline). This is achieved thr放習ough a multicom很信ponent system that incorpora間照tes active silencing by the tTS林窗 protein in the a視答bsence of tetracy就也cline and strong activation by th自商e rtTA protein in the presence 空遠of tetracycline. In the 畫城absence of tetracy媽相cline, the tTS protein derived from電用 the fusion of TetR (Tet repressor 書民protein) with KRAB-AB (the transcri玩但ptional represso讀報r domain of Kid-1 prote紙舊in) binds to the TRE promoter lea房低ding to the active suppression of gen物紅e transcription. The r數明tTA protein, on the other hand,作線 derived from the 拍子fusion of a mutant T少議et repressor to VP16 (the transcript兒計ion activator domain of virion prot通坐ein 16 of herpes simplex體時 virus), binds t間行o the TRE promoter to act睡亮ivate gene transcription o靜地nly in the prese我他nce of tetracycline.

While our standard pigg慢空yBac inducible gene expression vector e樹討xpresses tTS and rtTA as a飛地 fusion protein, which acts as a gen農動e activation switch, the l下廠ow leak version of綠電 our piggyBac Tet-On vector吃時 is designed to 匠樹allow tissue-specific indu計金ction of target transgenes in th費一e presence of tetra一放cycline while minimizing通要 leaky expression in她間 non-target tissu科綠es in the absence of te兒化tracycline. There are th動書ree expression cassettes in明術 this vector: 1) th和什e GOI driven by t拿弟he TRE promoter, 2)&n門金bsp;the tTS gene driven by a ub暗坐iquitous promoter, and 3) th見睡e rtTA gene dri遠城ven by a user-selected tissue-speci船舞fic promoter. In th日司e absence of tetracycline, the tTS 機對protein, ubiquitously expresse道對d in all tissues, bind些他s to the TRE promoter with high af北山finity thereby suppressing微個 the GOI express船個ion in all tiss拍開ues. In the presence of tetr空煙acycline, the rtTA 慢光protein, which is specifi火裡cally expressed i做樹n the target tis討分sue, can b姐就ind to the TRE promoter to activate書分 the GOI expression only in場會 the target tissue.

Our piggyBac inducible gene e兒煙xpression system comprises t愛農wo components: the 廠火transposon plasmid an在家d the transposase (h事站elper PBase). The transposon plasmid 那黑contains two terminal repeats (T北照Rs) bracketing the regio不北n to be transposed. 通厭For the low leak ver友飛sion of this vector, all three expre街年ssion cassettes consisting場吃 of the GOI, the tTS 雪裡gene, and the rtTA gene d多身escribed above are clone鐘很d into this region. The 區土transposase can be delivered into頻算 target cells through two methods. The 問湖helper plasmid can be transiently tr笑店ansfected into cell跳湖s. Alternatively, targ不都et cells can be injected with transp錢區osase mRNA generated光如 by in vitro trans街數cription from the 這也helper plasmid. When the helper資吃 PBase and the piggyB低東ac transposon vector are co-introduc你見ed into target cells, the t的日ransposase produced from the help藍國er would recogni長弟ze the two TRs on the transposon and數電 insert the flanked region i南來ncluding the two TRs i厭街nto the host genome. Insertion typic空個ally occurs at host chromosomal sites t分校hat contain the TTAA sequence些雪, which is duplicated on the two flank城去s of the integrated fragment. Gene e錢那xpression can then 什路be turned on in你學 the presence of tetracycline. 照樂Through both methods of de她紅livering transposase,也媽 it is expressed for on吃湖ly a short time. Upo做東n the loss of the helper plasmid or d妹站egradation of transp學人osase mRNA, the integration o業議f the transposon into the 爸刀host genome becomes perm事用anent.

PiggyBac is a class II計南 transposon, meaning綠校 that it moves in a cut-and木能-paste manner, hopping志看 from place to place without 做會leaving copies behind. (In cont錢舊rast, class I transpos動呢ons move in a copy-and-paste manner討國.) If the transp從年osase is reintr算兒oduced into the cells, the transposon歌錢 could get excised fro近制m the genome of some cells, foot公司print-free.

For further inf理和ormation about this vector system, p師北lease refer to the papers be音還low.

Limited cell type range: The delive下跳ry of piggyBac vectors into cel河相ls relies on transfe現靜ction. The efficiency 年低of transfection can vary greatly會慢 from cell type t西子o cell type. Non-divi民醫ding cells are of妹到ten more difficult微說 to transfect than dividing cells,費文 and primary cells are oft山道en harder to trans在笑fect than immortaliz文近ed cell lines. S地電ome important cell types, such a劇雪s neurons and pancreatic β cells, ar術費e notoriously difficult to tra問坐nsfect. Additionally, plasmid transfe員她ction is largely limited to廠火 in vitro applications a美內nd rarely used in vivo. These issues 票日limit the use of the pig弟長gyBac system.

5' ITR: 5' inverted terminal re得筆peat. When a DNA sequenc行開e is flanked by two I請刀TRs, the piggyBac transpose can r通從ecognize them, and insert t醫輛he flanked region inc亮她luding the two ITRs into the 靜音host genome. 

TRE: Tetracycline-responsive element p行校romoter (2nd generation).年討 This element can be regulated by又地 a class of transcription factors 笑體(e.g. tTA, rtTA and tTS)中民 whose activities are dependent on te家務tracycline or its analogs (e高見.g. doxycycline).

Kozak: Kozak consensus se他都quence. It is plac他國ed in front of the start窗那 codon of the ORF of interest to fac著我ilitate translation報些 initiation in eukaryotes.

ORF: The open reading f劇森rame of your gene 司對of interest is plac現著ed here.

rBG pA: Rabbit beta-globin 藍話polyadenylation sig呢科nal. It facilitates transcriptional議可 termination of the女術 upstream ORF.

Promoter: The promoter chosen to drive exp女算ression of the rtTA protein.

rtTA: Reverse tetracycline responsive transc男器riptional activator M2 (2街志nd generation). This protein binds t日這o TRE promoter to activate街從 gene transcription o什呢nly in the presence of tetr到一acycline or its analogs (e.g. doxycyc光冷line). It has high這些er sensitivity to the inducing drug and機鐵 lower leaky activity in the absence 體可of the drug compared to 的區its predecessor.

SV40 late pA: Simian virus 40 lat北校e polyadenylatio音劇n signal. It facilit關街ates transcriptional termination of th務小e upstream rtTA protein.

CBh promoter: CMV early enhancer fuse分司d to modified chicken β-a鐘很ctin promoter. This drives the exp器我ression of the downstream筆海 tTS protein.

tTS: Tetracycline-control員公led transcriptional silence秒船r. This protein 綠會binds to TRE prom關一oter to actively suppres上森s gene transcription很南 only in the absence of刀兵 tetracycline and its analogs (e.g.長年 doxycycline).

SV40 late pA:&n街爸bsp;Simian virus 40 late polyadenylati在生on signal. It facilitates transc又睡riptional termination of the 新河upstream tTS protein. 

3' ITR: 3' inverted terminal repeat.

Ampicillin: Ampicillin 習姐resistance gene. It allows the plasmi市森d to be maintai自畫ned by ampicillin selection請志 in E. coli.

pUC ori: pUC origin of r司友eplication. Plasmids carryi外照ng this origin exist in high copy n吃自umbers in E. coli.