PiggyBac FLEX Conditional G著呢ene Expression Vector (Cre-On)

The piggyBac FLE少船X conditional Cre姐好-On gene expression間請 vector combines VectorBuilder’好說s highly efficient樹著 piggyBac vector system with the C輛商re-responsive FLEX conditional gene e車知xpression system t紙老o help you achieve通我 transfection-m火地ediated permanent i放坐ntegration of Cre-res不說ponsive FLEX switch into the host和可 genome. The FLEX Cre-On swi呢見tch utilizes two pairs of LoxP-varia就章nt recombination sites flanking a船信 gene of interest in an arrangement t自城hat completely inhibits gene ex理但pression in the absence of Cr答樹e and activates ge音什ne expression upon Cre-depe也報ndent inversion of the codin見窗g sequence.

The FLEX Cre-On switch consists of tw什黑o pairs of hete放工rotypic LoxP-variant rec年少ombination sites, name熱門ly LoxP, having the wild-是長type sequence, an家唱d Lox2272, having a mutated問有 sequence, flanking an ORF whic著白h is in the reverse (anti可務sense) orientat數機ion relative to the pr不也omoter. Both LoxP variant制風s are recognized by坐銀 Cre, but only identical pairs of Lox小票P sites can recombin可從e with each other and not with any 化資other variant. The LoxP a志討nd Lox2272 sites are organize厭間d in an alternating 物睡fashion, with an antiparallel ori了相entation for each pair. In t樹議he absence of Cre recombinase, t做拍he ORF is not expressed 爸算due to its antisense 船間orientation relative to the promote就知r. In the presence of Cre, the 拍頻LoxP and Lox2272快刀 sites undergo recom藍跳bination with the other 會分LoxP and Lox2272 sites res線風pectively, resulting in the區去 inversion of the ORF to a sense orie書店ntation and excision o秒可f one from each pair of id頻紅entical recombination sites. Thi學友s allows the user-selected pro公門moter to drive the transcripti路個on of the gene of interest. Since the火報 ORF is now flanked by數和 two different LoxP-variant sites, 他她no further recombination events但公 will take place even when 個民Cre is present.

The piggyBac vector system is te鐵些chnically simple, utili吃兒zing transfection (rather tha學信n viral transduction) to pe請謝rmanently integrate your gene(s) 從鐵of interest into the host genome.&nb件子sp;The piggyBac FLEX conditio很票nal Cre-On gene expression sys自音tem comprises two componen鄉算ts: the transposon plasmid and the 慢唱transposase (helper 上藍PBase). The transposon p電物lasmid contains two termi小都nal repeats (TRs有費) bracketing the r短算egion to be transposed. The FLE路身X Cre-On switch describ這用ed above is cloned into thi些工s region. The transposase can be de體土livered into tar弟很get cells through two metho還就ds. A helper plasmid通志 encoding PBase can be transi少這ently transfecte人年d into cells. Alter媽看natively, target cells can be 南行injected with in vitro transcribed 兒匠PBase mRNA. When the helper PBase and 呢呢the piggyBac transposon vector are 很小co-introduced into target cells, th麗理e transposase produced from the helper什水 would recognize 嗎視the two TRs on the 到輛transposon and insert長雪 the flanked FLEX Cre-O討近n switch including the two他近 TRs into the host新費 genome. Insertio我紙n typically occu了通rs at host chromos森路omal sites that contain the TTAA seq關飛uence, which is duplicated 雨制on the two flanks 這坐of the integrated fra女樂gment. Gene expression can the綠光n be activated in the prese空信nce of Cre upon Cre-mediated invers新區ion of the coding sequence. 匠少Through both methods of deliverin懂議g transposase, it is e知場xpressed for onl子那y a short time.區討 Upon the loss of the h對小elper plasmid or degradation o秒我f transposase mRNA, the integr得很ation of the transpo南黃son into the host geno裡報me becomes perman睡現ent.

PiggyBac is a class II有器 transposon, meaning that it move我答s in a cut-and-paste manner, hopping作這 from place to place器員 without leaving copies behind音你. (In contrast, cl公報ass I transposons mo見信ve in a copy-and-paste manner.) If th公街e transposase is reintroduce生請d into the cells, the transposon could 呢筆get excised from the genome of some cel會通ls, footprint-free.

While this vector 笑錯system can be use廠黃d in tissue culture cells, it i舞冷s particularly suitable for the 體議generation of transgenic animals. Wh離放en a transgenic animal ca相銀rrying such a vec路廠tor system is crossed to水工 an animal carrying低腦 a tissue-specific Cre transgene, th美月e gene of interest w河飛ould be turned on in the progeny 吧外animals carrying bot見資h types of transgenes, speci作吃fically in cells where 遠市the tissue-specific Cre i那動s expressed and the知關 user-selected promoter driving the 藍很gene of interest is active.

For further information about this電一 vector system, please refer to the p雜現apers below.