Lentivirus Tet Inducible Gene Expres習亮sion Vector

VectorBuilder's lenti腦分virus inducible gene 木話expression vector combines th了從e highly efficient third什空-generation lenti看什viral vector system with the 農男Tet-On inducible gene expressio章問n system to help you achieve 車刀permanent integration of喝鐵 tetracycline-induc爸月ible gene expression ca短對ssettes into the host genome.

The Tet-On inducible gene 慢秒expression system is煙話 a powerful tool to control t懂哥he timing of expression of t輛些he gene(s) of interest (GOI) in mammali店街an cells. Our Tet-On inducib日河le gene expression vectors are designe森空d to achieve nearly complete sil嗎答encing of a GOI in the absence of te還熱tracycline and its analo老短gs (e.g. doxycy嗎公cline), and strong, rapid行讀 expression in r物樹esponse to the addition of tetracyclin南山e or one of its analogs (e.g. doxy區術cycline). This is a好區chieved through a multicomp票家onent system which incorporate村頻s active silenci河暗ng by the tTS protein in the a視森bsence of tetracycline些微 and strong activation by the rtTA pro拿子tein in the presence of t裡火etracycline. In the absence of你行 tetracycline, the tTS p吧答rotein derived from 城影the fusion of TetR (Tet repre計坐ssor protein) and KRAB-AB (the tra弟要nscriptional repres如科sor domain of Kid-1 pro事頻tein) binds to the tetracycline內地-responsive element (TRE) promoter,&nb金體sp;leading to the 房從active suppression of 購信gene transcription. The rtTA prot話北ein, on the other hand下吃, derived from the fusion of懂高 a mutant Tet repressor and VP1鐘件6 (the transcription act體司ivator domain of 歌雨virion protein 16 of herp在下es simplex virus), bi不暗nds to the TRE promote年術r to activate gene transcripti雜影on only in the pres文答ence of tetracycline.

The lentiviral vector學行 system is a highly efficient v謝計ehicle for introducing genes perman拿靜ently into mammalian cells. A lentivira票下l vector is first constructe視白d as a plasmid 中兒in E. coli. For the lentivirus Tet in會廠ducible gene expression ve喝白ctor, the tetracycli還老ne inducible expression cassette cons購技isting of the TRE promoter driv公海ing the GOI is placed in-betw鐘要een the two LTRs during vector constru醫答ction. It is then transfected int玩湖o packaging cells along with s吧路everal helper pl村吧asmids. Inside the packaging c長雜ells, vector DNA located是現 between the two long terminal repea歌區ts (LTRs) is transc頻請ribed into RNA, a到很nd viral proteins expressed by the hel體見per plasmids further packa報睡ge the RNA into virus. Live virus i在但s then released into the supernatant,些請 which can be used t東還o infect target cells dir要對ectly or after concentration.

When the virus is adde畫來d to target cells, the請慢 RNA cargo is shuttled into cell舞紅s where it is reverse transcribed火腦 into DNA and randomly integra什暗ted into the host genome. 火的The inducible expression casse車匠tte placed in-between the two LTRs 吧信during vector construction煙哥 is permanently inserted into 窗見host DNA alongside the rest of vi跳老ral genome. 

While our lentivirus Tet induc子事ible gene expression vector includes an舞吃 inducible gene expres雜有sion cassette consistin線器g of the TRE promoter drivin知我g the user-selected 長林GOI, the TRE binding regulat到唱ory proteins tTS and rtTA 紅區have to be provided using a separate外喝 helper vector t司這o achieve tetracycline induced gene exp森道ression in the pre國北sence of tetracycline,話靜 while minimizing leaky express姐冷ion in the absence of tetracycli街兵ne. For the len來和tivirus Tet inducible ge紅快ne expression vector喝會 system, the two-vector syst醫體em achieves higher levels of transgene醫筆 induction in the p務關resence of tetra術飛cycline compared to an all-i日跳n-one vector system. 但市An all-in-one vector consists of two 的舞consecutive expression c個長assettes: the GOI driven b多間y TRE promoter 街老and the tTS/rtTA genes dri個姐ven by a ubiquitous科劇 or tissue-specific promoter. Fo師裡r the lentiviral vectors, inter呢去nal polyadenylation signal is not s金司uggested to be pla呢笑ced between the LTRs for e議放ach individual expres跳電sion cassette, as this wou學視ld inhibit virus pac內討kaging. Instead, a sin些離gle polyadenylation藍林 signal is placed i是哥n the 3’LTR. As a result, trans了嗎cription from the upstream TRE pro我如moter often continues past the end 路一of the upstream ORF, through the 他視downstream promoters and O路美RFs (tTS/rtTA genes). This of相男ten leads to partial inhibition of和拍 expression of the do女道wnstream tTS/rtTA, th生鐵erefore preventing efficient inducti靜知on of gene expression in the prese爸醫nce of tetracycline. Ther歌媽efore, we recommend co年知-transducing target cells 就科with lentivirus carr呢雪ying the TRE driven GOI and lentivirus放靜 expressing the 如山tTS/rtTA cassett什空e to achieve the best i上醫nduction efficiency.

By design, lentiviral vecto弟用rs lack the genes required for viral pa答月ckaging and transduction (these genes a司下re instead carri國吧ed by helper plasmids used 和票during virus packaging)有對. As a result, v報紅irus produced from lent路金iviral vectors has the important s舞街afety feature of being replicati科門on incompetent (如人meaning that they can transduc廠新e target cells but cann金話ot replicate in th南動em).

For further information about thi的照s vector system, please refer to the 姐看papers below.