PiggyBac FLEX Co小西nditional Gene Expression Vector (鐘化Cre-Off)

The piggyBac FLEX conditiona朋愛l Cre-Off gene e裡下xpression vector com中木bines VectorBuilder’s highly efficie刀行nt piggyBac vector system with the C鄉話re-responsive F討鐘LEX conditional gene expr數這ession system to help y腦唱ou achieve transfection-mediated p頻刀ermanent integratio這愛n of Cre-respon水弟sive FLEX switch into the host genome.厭你 The FLEX Cre-Off switch utilizes t放水wo pairs of LoxP-可亮variant recombination sites flanki樹公ng a gene of interest in an arra白對ngement that facilitates ro校能bust inactivati他業on of gene expression b務兵y Cre-dependent 間醫inversion of the coding西報 sequence.

The FLEX Cre-Off switch consists of理討 two pairs of heterotypic LoxP-varian著舞t recombination site腦話s, namely LoxP having 花那the wild-type sequence and L要學ox2272 having a m農照utated sequence, flanking an ORF東妹. Both LoxP var子黑iants are recognized by Cre, b村拿ut only identical pairs of LoxP si要笑tes can recombine with each other 少冷and not with any othe妹鄉r variant. The LoxP and 章個Lox2272 sites are org暗爸anized in an alternati子區ng fashion, with an antiparallel ori機放entation for each pair. In the absenc亮間e of Cre recombinase,慢影 the ORF can be expressed under the con購路trol of the user-selected pro他醫moter. In the presence of Cre, th區數e LoxP and Lox2272 sites un那數dergo recombination with the other Lo通看xP and Lox2272 sites respectively, resu黑商lting in the inversio票電n of the ORF to an antisense和愛 orientation and excis好物ion of one from each pair of id刀了entical recombination sites慢動. Inversion of the ORF pre鐵還vents expression of the gene of費國 interest. Since the ORF is now秒裡 flanked by two different LoxP-個妹variant sites, no車日 further recombinat村多ion events will take p白員lace even when Cre is朋鐘 present.

The piggyBac vec亮但tor system is te門好chnically simple, utilizing transfectio草呢n (rather than viral trans暗店duction) to permanentl從就y integrate your gene(s) of in船體terest into the host genome. Th草內e piggyBac FLEX cond風兒itional Cre-OFF gene expre報海ssion system compris林國es two components: t短很he transposon pla綠小smid and the transposase (helper 朋知PBase). The transposon plasmid contai哥麗ns two terminal repeats (TRs) 那人bracketing the region t可答o be transposed. The FLEX Cre-O視雪FF switch described above is cloned in理章to this region. 紙還The transposase 了綠can be delivered into target cells thro還如ugh two methods. A helper plasmid物煙 encoding PBase ca小什n be transiently transfec的腦ted into cells. Alter兵雪natively, target cells can be injecte快他d with in vitro transcrib也師ed PBase mRNA. When the helper PBase樂小 and the piggyBac transposon 為玩vector are co-introduced into t資拿arget cells, the transposa間店se produced from the helper錯制 would recognize th道微e two TRs on the tran校人sposon and insert the flanke現能d FLEX Cre-OFF switch inclu畫來ding the two TRs into the host genome學票. Insertion typically occu匠頻rs at host chromoso要學mal sites that conta音筆in the TTAA sequence, which is duplic腦信ated on the two flanks of the integr爸相ated fragment. Gene expression和著 can then be inact好化ivated in the pr訊唱esence of Cre recombinase upon Cre-med算生iated inversion of the coding seq錯林uence. Through both methods of 海東delivering trans愛自posase, it is expressed f水光or only a short time. Upon the loss 草關of the helper plasmid 師花or degradation of tr街習ansposase mRNA, the i長兵ntegration of the transposon公區 into the host genome becomes per如拿manent.

PiggyBac is a class 短高II transposon, meaning tha鄉熱t it moves in a cut-很司and-paste manner, hopping from plac暗笑e to place without 熱鐵leaving copies behind歌唱. (In contrast, class I transposo高物ns move in a copy-and-paste mann姐也er.) If the transposase is動樂 reintroduced int報器o the cells, the transp老們oson could get excised from the genome有窗 of some cells, 南輛footprint-free.

While this vector 吧村system can be used in tissue cultur物站e cells, it is particularly suitable路微 for the generation of tran就個sgenic animals. When a transgen黃為ic animal carrying such a v些姐ector is crosse市房d with an animal 海的carrying a tissue-specific 森間Cre transgene, the ge算時ne of interest woul討上d be turned off in the progeny anima姐我ls carrying both types of transgene費錯s, specifically in cells報志 where the tissue-specific Cre is ex年風pressed and the user-selecte車飛d promoter driving the gene of 藍和interest is active.

For further information abo小這ut this vector 務制system, please refer to多森 the papers below.

5' ITR: 5' inverted terminal repeat. Wh們雨en a DNA sequence 藍草is flanked by two體秒 ITRs, the piggyBac tra西我nspose can recognize them, and insert t的新he flanked region includi草樂ng the two ITRs into the host gen他間ome.

Promoter: The promoter driving your gene of購小 interest is placed here.

Lox2272: Recombination site for Cre recomb謝廠inase. Mutated L吃白ox site with two base substitut們呢ions of wild type LoxP. Incompatible嗎村 with LoxP sites. When Cr近工e is present, the LoxP and LoxP2272 民錯sites will be cut and re國道combine with compatible sites.

LoxP: Recombination site f那銀or Cre recombinase. Incomp空議atible with Lox22師森72 sites. When Cre is 文睡present, the LoxP and L什煙ox2272 sites will be c愛線ut and recombine with compatibl從又e sites.

ORF: The open reading frame of your gene 鐘拿of interest is placed here,能東 in a sense orientation.

rBG pA:  Rabbit beta-globin polyadenylation sig暗信nal. It facilitates transcr離費iptional termination of the 這商upstream ORF.

CMV promoter: Human cytomegalovi可信rus immediate early pr拍木omoter. It drives the u來商biquitous expression of the 西著downstream marker gene.

Marker: A drug selection gene (suc我請h as neomycin res可近istance), a visually detect樂人able gene (such as EGF數煙P), or a dual-reporter gene (such as離拿 EGFP/Neo). This allows ce煙玩lls transduced with the vect科大or to be selected a會畫nd/or visualized自影.

BGH pA: Bovine growth hormone p化車olyadenylation signal遠到. It facilitates transcriptio有唱nal termination of the upstream OR紅報F.

3' ITR: 3' inverted terminal repeat.

Ampicillin: Ampicillin resistance 看公gene. It allows t從商he plasmid to be maintained by ampicil拿鐵lin selection in E. coli.

pUC ori: pUC origin of replication. P花照lasmids carrying this origin 妹問exist in high copy 照快numbers in E. coli.