Tol2 FLEX Conditiona玩紙l Gene Expression V小鄉ector (Cre-Switch)

The Tol2 FLEX conditional Cr件黃e-Switch gene expression v鄉費ector combines Vect分拍orBuilder’s highly effi服工cient Tol2 vector system with 黑綠the Cre-responsive FLEX 近答conditional gene ex近喝pression system to h她校elp you achieve transfection-med請車iated permanent integration of北習 FLEX switch int數照o the host genome 黑歌for Cre-induced switching bet多文ween the expression of two ORFs. The錯音 FLEX Cre-Switch sy動畫stem utilizes two pairs of LoxP-va拍那riant recombination sites flankin件慢g two antiparallel ORF子知s in an arrangeme輛時nt which facilitates activa船個tion of one gene while repressing th還對e other by Cre-depen為你dent inversion of both ORFs.

The FLEX Cre-Switch system consi做還sts of two pairs of heterotypic Lo費街xP-variant recombinatio很看n sites, namely拍南 LoxP, having the wild type sequence a報朋nd Lox2272, having a mutated sequence他機 flanking a pair of ORFs. Both Lox他自P variants are r信請ecognized by Cre,刀內 but only identical pairs of LoxP sites訊鐵 can recombine with each other and not樂我 with any other 光那variant. The two ORFs are in an opposit的湖e orientation with respect to 火說one-another, such that one妹答 ORF is in its 白分proper sense orientation,低妹 while the other is in an an內訊tisense orientation. The LoxP an舊兒d Lox2272 sites are org會答anized in an altern是理ating fashion, with an 員東antiparallel orientat但線ion for each pair. In the absence of C錯錢re recombinase, while the first ORF is 厭科expressed under the control of t離金he user-selected promoter, 站村the second ORF is not expressed麗鐵 due to its antisense orientation.森影 In the presence 術音of Cre, the LoxP and Lox2272 si舊謝tes undergo recombination with the 女書other LoxP and Lox2272 sites resp業錢ectively, result購在ing in the inversion of both 你草ORFs and excision裡睡 of one from each pair of id自照entical recombinati費看on sites. Inversion of the ORFs results秒工 in silencing of the f理會irst ORF (which will now be in an訊亮 antisense orient聽土ation) and allows exp煙會ression of the second車器 ORF (which will now你討 be in a sense orientation).

The Tol2 ve雨水ctor system is technically simp房哥le, utilizing transfection (水長rather than viral transduct兒亮ion) to permanently integrate your 城亮gene(s) of interest into t裡黑he host genome. T可們he Tol2 system comprises tw謝計o components: the transposon plasmid熱開 and the transposase.站開 The transposon plasmid contains two i書說nverted terminal r票計epeats (ITRs) bracketing the region to但弟 be transposed. The FLEX Cre-Switch de科務scribed above i哥拍s cloned into this region. 書還The transposase can be delivered能店 into target cells through 雪機two methods. The helper麗錯 plasmid can be transiently tra飛人nsfected into cells土問. Alternatively, target ce嗎腦lls can be injected 匠拿with transposase mRNA generated by i秒間n vitro transcription from the helpe視暗r plasmid. When the tr男山ansposon and helper 雪站plasmids are co-transfected into tar輛快get cells, the transposase produce能新d from the helper plasmid woul多低d recognize the two ITRs on the tr司遠ansposon and insert the fl個書anked region including the two ITRs int離問o the host genome. At each insertion si朋技te, the Tol2 transpos多廠ase creates an 8 bp開費 duplication, resulting in ident志站ical 8 bp direct repeats fl如姐anking each transposon 討花integration site in the genome.家章 Insertion occurs with空兒out any significant bias for t友小he insertion site sequence. T森理his is unlike transposon system討答s which have speci外花fic target consensus sit空紅es. For example, piggyBac trans裡讀posons are typically ins吃哥erted at sites containing the seque問答nce TTAA. Expression of t音腦he second ORF in the FLEX Cre-樹但Switch can then be 件對activated while silencing the first 舊紅ORF in the presence of Cre recomb快習inase, upon Cre-media知和ted inversion of bo身醫th ORF sequences. Through both methods車火 of delivering transposase, it is expre愛又ssed for only a shor吃家t time. Upon th房東e loss of the helper p要外lasmid or degrada子不tion of transpo廠快sase mRNA, the integration of the tra嗎北nsposon into th到兵e host genome becomes permanen議得t.

Tol2 is a class II transposon,弟公 meaning that it moves in a謝歌 cut-and-paste manner, hopping樹風 from place to place without leaving co黑訊pies behind. (In contrast, class I 費電transposons move in a copy農場-and-paste manner.鐘公) If Tol2 transpos區妹ase is reintrodu日湖ced into the cells, the transposon coul有醫d get excised from the genome 風歌of some cells, resulting in either prec些吧ise or imprecise excisions 近遠with indels created.

While this vector system劇相 can be used in tissue culture cells, i低湖t is particularly suitable for t訊一he generation of transgen明見ic animals. Transgenic animals carrying懂土 such a vector originally 朋我express the first user-sel公章ected ORF, however when crossed物見 to an animal carrying 窗生a tissue-specific Cre transgene, ex開的pression of the second user-selected O飛東RF will be activated while silencing近要 the first ORF in the progeny animals 請姐carrying both types of 機亮transgenes, specifically in cells wh間開ere the tissue-specific Cre is express多船ed and the user-selected promote畫嗎r is active.

For further informa算物tion about this vector system, pl森嗎ease refer to the papers below場是.

The Tol2 FLEX c動大onditional Cre-Switch gene expres科錯sion vector is designed to achieve 章為Cre-mediated switching betw他什een expression of物到 two ORFs in mamma聽腦lian cells and anim什有als. Expression is und上湖er the control of a user-sel黑房ected promoter&綠睡nbsp;and can be permanently switched 廠錯from one user-selected ORF to another 裡動by co-expression 知日of Cre recombinase. 

This vector along with t電店he helper plasmid en人鄉coding the Tol2 鐵工transposase are optimized for high cop城舞y number replication in E. coli, e年上fficient transfection into a wide ran你謝ge of target cells, 得員and high-level expression of the tr門吃ansgene carried on the vector事年.

Limited cell type range:&nb問見sp;The delivery of Tol2 vectors into cells服明 relies on transfection. The ef但是ficiency of transfection c可關an vary greatly from cell type to ce電文ll type. Non-dividing cel唱市ls are often more difficult to 司那transfect than dividing cell舊站s, and primary cel就現ls are often harder to transfect畫票 than immortalized cell li民算nes. Some important cell types看火, such as neurons 輛南and pancreatic β cells, are notorious通你ly difficult to transfect. T友樹hese issues limit the us術南e of the Tol2 syst聽低em.