PiggyBac FLEX C討車onditional Gene Expressio著照n Vector (Cre-Switch)

The piggyBac FLE服湖X conditional Cre-Switch 習地;gene expression vector&nb員市sp;combines VectorBuilder’s林拍 highly efficient 姐跳piggyBac vector sy讀動stem with the Cre-responsive F熱術LEX conditional gene expression sys鐵爸tem to help you achieve t就雨ransfection-mediated permane愛城nt integration of FLEX 森但switch into the host genom小器e for Cre-induc關分ed switching between the expressio那跳n of two ORFs. The FLEX Cr月現e-Switch system utilize媽上s two pairs of 分亮LoxP-variant recombi拍去nation sites fla我秒nking two antiparallel ORFs in an商老 arrangement which facilitates act場員ivation of one gene while吧厭 repressing the other by Cre家拍-dependent inversion of both ORFs.

The FLEX Cre-Swit在紙ch system consists of two pairs of h從紙eterotypic LoxP-var小公iant recombinati遠化on sites, namely LoxP, havi笑什ng the wild-type sequen唱南ce, and Lox2272, having a場從 mutated sequence flan身現king a pair of ORFs. Both LoxP variants綠小 are recognized by C又照re, but only identical pairs of Lox電吃P sites can recomb火南ine with each othe來那r and not with any other variant. The t遠算wo ORFs are in an opposite orientation視木 with respect to錢刀 one another, su姐鄉ch that one ORF 文木is in its proper sense orientation, wh房會ile the other is in an antisense or低身ientation. The LoxP and Lox2272 site車雨s are organized in an alternatin西下g fashion, with an antiparallel or師木ientation for each pair. In the absen信是ce of Cre recombinase, while the first 區店ORF is expressed under the con樹資trol of the user-selected promot但用er, the second ORF is 學朋not expressed due to its antisense o還可rientation. In the presen現弟ce of Cre, the LoxP and Lox2272 si睡很tes undergo recom要也bination with the ot務銀her LoxP and Lox2272 s體長ites respectively, resul訊呢ting in the inversion of both ORFs a長對nd excision of one from each p水南air of identical recombination 銀筆sites. Inversion of t綠購he ORFs results in silencing of店能 the first ORF (which will now書厭 be in an antisense orientatio雨動n) and allows expression of th的外e second ORF (which will now be 山雨in a sense orientation).

The piggyBac vector 空紙system is technically simple, utilizi作兵ng transfection (rather than viral tran火公sduction) to permanently integrate道上 your gene(s) of 相還interest into the host genome. 來生The piggyBac FLEX conditional什金 Cre- Switch gene exp場答ression system comprises two compo照劇nents: the transposon謝亮 plasmid and the transposase (helper 吃上PBase). The transposon plasmid con火時tains two terminal r金光epeats (TRs) bracketing the regi弟工on to be transposed. The FLEX Cre-S綠微witch described above白近 is cloned into this region. The tra鐘見nsposase can be deliver多紅ed into target cells 海街through two method議唱s. A helper plasmid enc海明oding PBase can be t的草ransiently transfected into cells. Alte事秒rnatively, target cells 開廠can be injected with in vitro 的木transcribed PBase mRNA. When the hel讀如per PBase and the piggyBac tran妹雜sposon vector are co-introduced into ta身煙rget cells, the transposase produced 理又from the helper would recognize明城 the two TRs on the transposon a月們nd insert the flanked FLEX Cre-Swit書銀ch including the科件 two TRs into the host我開 genome. Insertion內如 typically occurs at host chromo和場somal sites that con商答tain the TTAA sequenc南快e, which is duplicated on the two fl事生anks of the integrated 說算fragment. Expression of the second O術都RF in the FLEX Cre-Switch can計媽 then be activated while 哥慢silencing the first ORF 老習in the presence of Cre recombinas書通e, upon Cre-mediated inver動但sion of both ORF sequences. Throu信多gh both methods of頻影 delivering tra拍藍nsposase, it is expressed for only a 廠爸short time. Upon the loss of the 從體helper plasmid or degra女大dation of transposase mRNA, the in輛哥tegration of the transposon into the通場 host genome becomes perm短高anent.

PiggyBac is a class II tran也睡sposon, meaning that it m明你oves in a cut-and-paste manne也腦r, hopping from place to place w刀黑ithout leaving copies behind物車. (In contrast, class I tran對綠sposons move in a copy-and-pas間窗te manner.) If the trans得什posase is reintroduced into the跳用 cells, the transposon could get exci刀的sed from the genome of some cells來說, footprint-free.

While this vector system can be used i女樂n tissue culture cells, it i腦通s particularly suitable厭低 for the generation of transgenic作紙 animals. Trans輛從genic animals carrying such a vec用電tor originally expre音還ss the first user-文場selected ORF, however when crosse線事d to an animal carrying a tissue我來-specific Cre transgene, expression of 體人the second user-sel討南ected ORF will be activated while sile跳長ncing the first ORF in 是購the progeny animals carryin一時g both types of transgenes, sp土輛ecifically in cells where the tissue-sp業關ecific Cre is expressed, and the use森校r-selected promoter is active高校.

For further information about this 玩窗vector system, please refer to答土 the papers below.

Switch-like regulation: Opposite orientation of t子輛he two ORFs ensures that while t農科he ORF in the sense 熱船orientation is express麗嗎ed, the ORF in the些長 antisense orientation is repr業又essed without any leaky gen舞廠e expression.

Permanent integ土草ration of vector DNA:錢門 Conventional transfecti錯銀on results in almos看匠t entirely transien中西t delivery of DNA into host cells due 身兵to the loss of 議農DNA over time. This proble紙妹m is especially prominent in rapidly di信熱viding cells. In c煙子ontrast, transfec快家tion of mammalian cells with the pig作聽gyBac transposon plasmid along wi下謝th the helper plasmid can delive對子r genes carried on 森村the transposon permanently暗中 into host cells due to 謝紅the integration of the trans相兒poson into the host geno美西me.

Technical simplicity:問有 Delivering plasmid vectors into c又站ells by convent白視ional transfect唱西ion is technically個是 straightforward, and 票討far easier than virus-based vec空拿tors which require 少還the packaging of live virus.

Very large cargo space媽如: Our transposon vector呢著 can accommodate ~30 k廠船b of total DNA. The plasmid b森為ackbone and tran水但sposon-related sequences on靜坐ly occupies about 3.1 kb, 廠麗leaving plenty of room to accommod東慢ate the user's sequence of interest.