Tol2 FLEX Conditional Gene E生在xpression Vector (Cre小會-Off)

The Tol2 FLEX conditional&nb中書sp;Cre-Off gene expressio要不n vector combines VectorBuild唱我er’s highly efficient 物討Tol2 vector system 外體with the Cre-responsive FLEX conditio雨中nal gene expressio術朋n system to help you achieve transfe國土ction-mediated permanent integr海道ation of Cre-responsive FLEX switc唱也h into the host geno從綠me. The FLEX Cre-Off道白 switch utilizes two pairs of LoxP-var空高iant recombination sites fl聽說anking a gene of interest in an arran文友gement which facilitate笑玩s robust inactiv雪也ation of gene expre亮鐘ssion by Cre-dependent inversion of t機外he coding sequen草森ce.

The FLEX Cre-Of愛又f switch consists算還 of two pairs of he身算terotypic LoxP-variant recombination s男務ites, namely LoxP having the w著議ild-type sequence and Lox2272 having a明雪 mutated sequence, flan亮看king an ORF. Both LoxP variants 個愛are recognized 市們by Cre, but only ident相畫ical pairs of LoxP sites can recombin金森e with each other and not 通說with any other variant. The LoxP an一服d Lox2272 sites are organized鐘道 in an alternating fashion, with a鄉街n antiparallel orientation for each pa唱北ir. In the absence of Cre recombin很關ase, the ORF can be expressed under t舊對he control of the user-sel算現ected promoter. I月場n the presence of Cr電信e, the LoxP and Lox2272 sites 章短undergo recombination with the othe學公r LoxP and Lox2272 sites respective要生ly, resulting in the inv紅美ersion of the ORF to an antisense船裡 orientation and excisi又熱on of one from each pair of 明內identical recombination sites. Inversi吧自on of the ORF prevents expres愛兵sion of the gene 話知of interest. Since the ORF is now fl時雪anked by two different LoxP-vari煙業ant sites, no further recombination雨工 events will take p白離lace even when Cre is pr慢錯esent.

The Tol2 vector system is t少靜echnically simple, utilizing tran吧嗎sfection (rather than viral tran市話sduction) to permanently integr月兒ate your gene(s) of i學分nterest into the host genome.&nb朋說sp;The Tol2 system comprise時時s two component公器s: the transposon plasmid an做舊d the transposase. The 門到transposon plasmid contains 拍又two inverted terminal聽了 repeats (ITRs) bracketing唱長 the region to be tran件風sposed. The FLE那著X Cre-Off switch described above is clo兒空ned into this region. Th通醫e transposase ca子路n be delivered into target cells throu會空gh two methods. The 物白helper plasmid can be transiently tra視短nsfected into cells. Alternatively,銀就 target cells can be injected wit子地h transposase mRNA g我拿enerated by in vitro 鄉會transcription from the helper自通 plasmid. When the trans嗎信poson and helpe場我r plasmids are co-transfected into你器 target cells, the transposa算樂se produced from the helper plasmid wou文花ld recognize the 人算two ITRs on the transposon and insert t校什he flanked region includi微費ng the two ITRs into the host gen行制ome. At each insertion site, t兒分he Tol2 transposase creates an 8 bp 少技duplication, resulting 兒火in identical 8 bp direct repeats fl的如anking each transposon integr知姐ation site in the g信話enome. Insertion occur我吧s without any significant bias for學高 the insertion site sequ老飛ence. This is unlike transposon syste草土ms which have specific t討工arget consensus site音靜s. For example, piggyBac trans會船posons are typically inserted at site金請s containing th通慢e sequence TTAA. Gene expression can 生也then be inactivated in the prese下遠nce of Cre recomb錢窗inase upon Cre-m動匠ediated inversi能歌on of the coding sequence. Through b去電oth methods of delivering頻離 transposase, it is expressed 商從for only a shor裡呢t time. Upon the loss of the helpe商藍r plasmid or degradation of 國得transposase mRNA, the內習 integration of the transposon 些河into the host genom為電e becomes permanent.

Tol2 is a class II土報 transposon, meaning that it m西業oves in a cut-an西亮d-paste manner, hopping from p短廠lace to place without 報習leaving copies behind. (In contr金空ast, class I transposon制水s move in a copy-and-paste 遠購manner.) If Tol2 transposase is r愛亮eintroduced into th物議e cells, the transpo信畫son could get excised from the gen務金ome of some cel不問ls, resulting in either precis視生e or imprecise excisions with indels cr厭紙eated.

While this vector s我朋ystem can be used in tissue遠離 culture cells, it is particularly sui多妹table for the generation of tra些鄉nsgenic animals. When a transgenic a物離nimal carrying such a vector is cros愛錢sed to an animal carryin件我g a tissue-specific Cre tra喝離nsgene, the gene of interest wo機市uld be turned off in the progeny機草 animals carrying both type城紅s of transgenes, specifica費司lly in cells where the tissue-spe好制cific Cre is expressed and the 我的user-selected promote理農r driving the gene of interest is a雨子ctive.

For further information abo什理ut this vector system, please refe少長r to the papers below.

Switch-like gene inactivation: Inversion of the user-select自動ed ORF to its antisense orientation i舊公n the presence of Cre 聽熱recombinase prevents any le藍金aky gene expression.

Stable gene inactivation房空: Treatment with Cre recombinase w外腦ill permanently invert the user-select商舊ed ORF to its antisense orien新舊tation. Upon invers頻微ion of the ORF to its antisense or身雜ientation followed by exci南北sion of one from each pair o下從f similar LoxP sites by recombination站謝, the ORF will be flanked by t黑玩wo different LoxP-variant sit弟謝es which will prevent further車國 recombination events even whe鐘多n Cre is present. This will permanen自生tly prevent transcriptio問畫n of the gene of interest.

Permanent integration of vecto懂子r DNA: Conventional tran們校sfection results in almost entirely人鐵 transient delivery of D坐資NA into host cells 討他due to the loss of DNA over tim師子e. This problem is esp女老ecially prominent in rapidl明學y dividing cells. In co我離ntrast, transfection of ma快煙mmalian cells with the Tol2 transposon 線錯plasmid along with the hel水老per plasmid can d要照eliver genes carried on the tra是訊nsposon permanently into host c師快ells due to the就離 integration of the transposon i化作nto the host genome.如媽

Technical simpl時們icity: Delivering plasmid vectors i外看nto cells by conventiona分醫l transfection is technic哥友ally straightforward, and far eas筆花ier than virus-based vectors現草 which require the packaging o友器f live virus.

Very large cargo s麗術pace: Our Tol2 關到FLEX conditional C河費re-Off gene expression v朋兵ector can accomm舊身odate ~11 kb of total DNA. The pla著吧smid backbone, transpos門唱on-related sequences and the sequen刀刀ces necessary for Cre-mediated爸厭 recombination only occupy about 3土關.1 kb, leaving plenty of 冷們room to accommodate the 刀器user's sequence of interest.

Limited cell type range: The delivery of Tol2 vectors in吧機to cells relies on transfection. The ef和鐵ficiency of tra靜村nsfection can vary greatly from cel雪冷l type to cell ty雜說pe. Non-dividing ce好票lls are often more difficult to 山工transfect than dividing cells, an視農d primary cells 吃電are often harder to transf火和ect than immortalized cell lines. Some一器 important cell types, s票嗎uch as neurons and pa對煙ncreatic β cells, are notoriou來視sly difficult to tra廠友nsfect. These issue司拿s limit the use of 笑厭the Tol2 system.

5' ITR: Tol2 5' terminal repeat. When a DNA行就 sequence is flanked吃風 by two ITRs, the Tol2 tr亮場ansposase can recog到鐘nize them, and ins紅靜ert the flanked region including t船答he two ITRs into the h有高ost genome.

Promoter: The promoter drivi是懂ng your gene of interest is pl師我aced here.

Lox2272: Recombination site for Cre recom小線binase. Mutated Lox site with玩但 two base subst但事itutions of wild ty志森pe LoxP. Incompa這雜tible with LoxP 老東sites. When Cre is presen為舞t, the LoxP and LoxP2272 sites w多請ill be cut and recombine件務 with compatible sites.

LoxP: Recombination著司 site for Cre recombinase.老車 Incompatible with Lox2272 sites. W船業hen Cre is present, the 的但LoxP and Lox227船村2 sites will be cut and recombine鐘農 with compatible sites.玩飛

Kozak: Kozak consensus seque兒舞nce. It is placed in fr我師ont of the start codon of the ORF 小快of interest because it is belie要房ved to facilitate t一很ranslation initiation in eukaryotes.

ORF: The open re文用ading frame of your gene o南嗎f interest is placed he西公re, in a sense orientation.

SV40 late pA: Simian virus 40 late po鄉到lyadenylation signal. It facil微煙itates transcriptional termi南線nation of the upstrea會年m ORF.

3' ITR: Tol2 3' terminal repeat.

Ampicillin: Ampicillin resistance gene. It al些國lows the plasmid to be maintained 資雜by ampicillin s習作election in E. coli.

pUC ori: pUC origi跳的n of replication. 能女Plasmids carrying讀森 this origin exist in high copy nu嗎輛mbers in E. col老舞i.