PiggyBac Tet Inducible Gene還個 Expression Vector

VectorBuilder’s piggyBac Tet inducible謝朋 gene expression習聽 vector combines the highly ef討到ficient PiggyBac vector sy機遠stem and the Te厭紅t inducible gene expression system t章人o help you achieve tran見從sfection-based perm玩做anent integration o樂你f tetracycline-inducible ge窗人ne expression cassettes into the 現低host genome.

The Tet-On inducible system is a po資村werful tool to cont書道rol the timing of expression o得化f the gene(s) of intere和站st (GOI) in mammal能議ian cells. Our T錢書et-On inducible gene expr民能ession vectors are designed to ach村票ieve nearly complete silencing of a G就放OI in the absence什南 of tetracycline and its analogs能現 (e.g. doxycycline), and str術黃ong, rapid expression in response to t得章he addition of tetracyclin這中e or one of its analogs (e窗為.g. doxycycline).器愛 This is achieved throug電報h a multicomponent system工時 that incorpora志有tes active silencing by th體化e tTS protein in拿秒 the absence of tetr金玩acycline and strong activation by th來議e rtTA protein in the pre業綠sence of tetracycline報女. In the absence of tetra算爸cycline, the tTS pr歌小otein derived from the fusion of Te黑民tR (Tet repressor protein) 鐵火and KRAB-AB (the transcriptional rep慢討ressor domain of Kid-1 protei朋老n) binds to the tetracy上輛cline-responsive element (TR物通E) promoter, leading to the active supp我多ression of gene transcription. The r廠紅tTA protein, on the other議服 hand, derived from the fusion of a m了很utant Tet repress跳民or and VP16 (the transcript我關ion activator d湖時omain of virion protein 16 of坐章 herpes simplex virus), binds to th對火e TRE promoter to activate gene transc村老ription only in the presence of tetra答在cycline.

While our piggyBac 媽師Tet inducible gene expression vector i公個ncludes an induc煙到ible gene expression cassette consisti場裡ng of the TRE promoter dri厭雜ving the user-selected GOI, the TRE 業哥binding regulatory proteins秒錯 tTS and rtTA must be pro務光vided using a se影中parate helper vector to ac線鄉hieve tetracycl間金ine-induced gene expressi又兵on in the presence跳爸 of tetracycline, wh近鄉ile minimizing leaky expression 照銀in the absence of tet時嗎racycline. 

Our piggyBac ind拍又ucible gene expression s麗高ystem comprises two components: th他來e transposon plasmid and the transpos匠好ase (helper PBase). The trans車計poson plasmid contains two terminal re土從peats (TRs) brac著影keting the region to be transpos動風ed. The tetracycline-i弟畫nducible gene expression cassette信近 consisting of the TRE promoter dr還那iving the user-selected GOI is一還 cloned in between the two TRs. The t土月ransposase can be delivered in睡街to target cells through two 身數methods. The helper plasmi紙微d can be transiently transfected into c議讀ells. Alternatively, ta跳南rget cells can be injected with t什來ransposase mRNA genera有會ted by in vitro t紙議ranscription from the helper plasmid. 銀但When the helper PBase爸風 and the piggyBac transposon vector ar資做e co-introduced into target cells, 年很the transposase produced f厭能rom the helper would recog飛房nize the two TRs on the tr河知ansposon and insert the flanked reg農你ion including t懂光he two TRs into th空學e host genome. Insertion們通 typically occurs at host ch老文romosomal sites that cont現著ain the TTAA sequence, whi長有ch is duplicated on the two flanks o男兒f the integrated fragment. Gene e跳習xpression can then兒鐵 be turned on in the presence of the近他 regulatory prote厭件ins rtTA and tetracycline我一. Through both m購熱ethods of delivering transposase, i拍車t is expressed for only a 船亮short time. Upon the紙藍 loss of the helper plasmid or deg白見radation of transposase mRNA, the inte不筆gration of the transposon into 問亮the host genome becomes perm資個anent.

PiggyBac is a class II tran低北sposon, meaning that it mo頻老ves in a cut-and-paste manner, hopping 筆村from place to place witho跳機ut leaving copi制地es behind. (In contras市件t, class I transposons move in a copy工機-and-paste manner匠舊.) If the transpos些火ase is reintroduced into the cells愛裡, the transposon could get 好劇excised from the genome of some cells,船章 footprint-free.

For further information abo銀科ut this vector s劇都ystem, please refer to the pa什文pers below.

Our piggyBac Te這生t inducible gene expression ve靜劇ctor when coexpressed with the Tet秒懂 regulatory protei信弟ns tTS and rtTA can achieve near東匠ly complete silencing of th金多e GOI in the absenc道房e of tetracycline, and strong, rapid e友水xpression in response t風土o the addition of 雪影tetracycline. Th錢作e piggyBac Tet ind煙錯ucible gene expression vector along wit鐵大h the helper plasmid are 討老optimized for high copy number replica火黃tion in E. coli, efficient tran樂電sfection into a wide rang海朋e of target cells, and hi技視gh-level expression of the transgene c員唱arried on the ve跳對ctor.

Limited cell type 討低range: The delivery of piggyBa章愛c vectors into cells rel媽煙ies on transfection. The efficie快冷ncy of transfection can vary gre舞亮atly from cell type to cell type校嗎. Non-dividing cells are often more 內可difficult to tra個科nsfect than dividing cells, and 購了primary cells ar拍城e often harder to transfect 計村than immortalized cell lines. Some 他關important cell types, such as neuron嗎離s and pancreatic β cells, are no微計toriously difficult to transfect子河. Additionally, pl志嗎asmid transfection is largely limite北劇d to in vitro applic快費ations and rarely used in vi舞遠vo. These issues靜體 limit the use of the pig視員gyBac system.

5' ITR: 5' inverted terminal repeat. Wh到吃en a DNA sequence is flanked by tw電匠o ITRs, the piggyBac transpose can reco要線gnize them, and insert the flanked reg少光ion including the two ITRs into the h媽黑ost genome. 些呢;

Promoter: The TRE promoter 友玩driving your gen家哥e of interest is placed here. 

Kozak: Kozak consensus sequen錯樂ce. It is placed in front 店飛of the start codon of the O也快RF of interest t有兵o facilitate translat女來ion initiation in eukaryotes.

ORF: The open reading frame 信又of your gene of interest is p生雪laced here.

rBG pA: Rabbit beta-globin polyadenylation s短林ignal. It facilitates transcri店用ption terminatio光技n of the upstream ORF.

CMV promoter: Human cytomegalovirus i村但mmediate early p志了romoter. It drives the ub音生iquitous expression of 紙開the downstream marker gene.姐現

Marker: A drug selection gene (s高花uch as neomycin resistance), a visuall好報y detectable gene (such as E自北GFP), or a dual-reporter gene (such as們喝 EGFP/Neo). This allows cells transfec鐘相ted with the vector to be sel空她ected and/or visualiz吃區ed.

BGH pA: Bovine growth hormone polyadenylation s吃制ignal. It facilitates亮服 transcriptional ter志報mination of the upstream ORF.

3' ITR: 3' inverted terminal repea兵兵t.

Ampicillin: Ampicillin resistance ge市得ne. It allows the雨長 plasmid to be maintained by amp信術icillin selection 年自in E. coli.

pUC ori: pUC origin of replica窗木tion. Plasmids carrying this orig黑愛in exist in high copy numbers in黑愛 E. coli.