Lentivirus Tet Regulatory 很看Protein Expression Vector

VectorBuilder’s lentivirus Tet regulat商年ory protein expression vector can be us如唱ed for lentiviral transduction-based 但章permanent delive請書ry of Tet regulatory proteins 照裡(tTS, rtTA, etc.) into mamm著地alian cells to help you achieve tetracy高筆cline-inducible exp秒間ression of target genes placed do遠家wnstream of a tetracycline resp數姐onsive-element (TRE了購) promoter.

The Tet-On inducible system is a 線說powerful tool to control the ti他關ming of expressi能日on of the gene(s) of interest (GOI) in可上 mammalian cells. Our 從不Tet-On inducible gene expression vector上都s are designed to achieve near店動ly complete silencing of a GOI in的歌 the absence of tetra算玩cycline and its anal裡去ogs (e.g. doxycycline), and strong, r短理apid expression in response to the ad討市dition of tetracycl山裡ine or one of its an喝拿alogs (e.g. doxycycline). This is 亮這achieved through a mult哥海icomponent system which incorpor視玩ates active silencing b兵大y the tTS protein in the absence of 自樹tetracycline and strong acti去暗vation by the rtTA protein 飛刀in the presence of tetracycline. In the紙公 absence of tetracycline, the tTS p少開rotein derived from the fusion高暗 of TetR (Tet repress商銀or protein) and KRAB-AB兵司 (the transcriptional repressor d刀門omain of Kid-1 protein) bin路習ds to the TRE prom了低oter, leading to the active sup志聽pression of gen姐報e transcription. The rtTA pro河船tein, on the other hand, deri歌們ved from the fusion of美人 a mutant Tet repr在林essor and VP16 (the t知離ranscription activator domain of v們知irion protein 1服們6 of herpes simplex virus)報微, binds to the TRE promoter to 短農activate gene transcription only in t厭術he presence of tetracyc冷也line.

The lentivirus Tet regulatory pr街些otein expression vector is first 信服constructed as a plasmid i生友n E. coli. The Tet regul內間atory protein expression cassette co慢討nsisting of the Tet regulatory prote妹務in(s) driven by懂農 a user-selected 還件promoter is placed in-between the two 唱這long terminal repeats (L什章TRs) during vector construction. I拿人t is then transfected市村 into packaging cells along w光弟ith several helper p視要lasmids. Inside the pack樂哥aging cells, ve不從ctor DNA located between two L通飛TRs is transcribe裡鐵d into RNA, and viral protei了但ns expressed by the helper p兒物lasmids further package the RNA i照暗nto virus. Live 來北virus is then release看行d into the supernatant, 土光which can be used to inf歌木ect target cells direc和雪tly or after concentration.

When the virus is added to target cell吧一s, the RNA cargo is shuttled into 雨問cells where it is reverse transc從銀ribed into DNA and randomly inte公子grated into the 吃靜host genome. The Tet regul鐵煙atory protein expressio爸放n cassette placed in-between the 術海two LTRs is permanen做著tly inserted into host DNA alongs歌個ide the rest of viral都河 genome.

While our lentivirus Tet regulatory p我快rotein expression vect到歌or includes an expression cassette co知業nsisting of the Tet regulatory protein森市(s) driven by a user兒紅-selected promoter, th現厭e GOI driven by the TRE p信著romoter must be pro近少vided using a separate helpe員小r vector to achieve tetracycline induc一術ed gene expression in the 窗聽presence of tetracycline,資山 while minimizing leaky expression i坐為n the absence of tetracyclin在關e.

By design, lentiviral vec視電tors lack the genes required for vi習自ral packaging and transduc理員tion (these genes are ins亮來tead carried by helper plasmids u來下sed during virus packaging). As a風樂 result, virus produced from lentiviral錯風 vectors has the important能站 safety feature of being replicati服水on incompetent (meaning t鄉到hat they can transduce target cells b雨聽ut cannot replicate in them).西中

For further information about th跳北is vector system, please refer to the 街關papers below.

Limited cargo space: The wildtype lentiviral 車要genome is ~9.2 kb. 請物In our vector, the compon兒笑ents necessary for viral packagin煙河g and transduction occupy ~2行懂.8 kb, which leaves ~6跳會.4 kb to accommodate the user’跳東s DNA of interest. Whe讀雜n the vector goes beyo們長nd this size limit, viral titer朋鄉 can be severely reduced. The lent道山ivirus Tet regulatory protein車水 expression vector is routi外到nely used for inserting s道暗everal functional elements besides th報影e ORF of the Tet regulatory慢近 protein(s), such as爸影 the promoter and dr湖下ug resistance cassette. If睡雨 the Tet regulat懂答ory protein(s) ORF 還少plus these additional elements exceed 6吧玩.4 kb, it could result 報物in compromised virus product分子ion.

Technical complexit森電y: The use of lentiviral vectors 兵費requires the production of live virus i去女n packaging cells followed員慢 by the measurement行用 of viral titer. These procedu西區res are technically deman吧玩ding and time cons空窗uming relative to conventional plasmid 都煙transfection.