Tol2 FLEX Condit船制ional Gene Expression Ve唱腦ctor (Cre-On)

The Tol2 FLEX conditional&n人廠bsp;Cre-On gene expressi嗎中on vector combines煙新 VectorBuilder’s high動山ly efficient Tol2 vector sy來銀stem with the Cre-responsiv呢個e FLEX conditional gene expression s書白ystem to help you achieve transfect高議ion-mediated permane議討nt integration of化但 Cre-responsive FLE內慢X switch into the host genome村雪. The FLEX Cre-On switch utilizes two 飛能pairs of LoxP-variant recomb冷輛ination sites f資校lanking a gene of interest in an 費呢arrangement which completely inhibi林刀ts gene expression 開女in the absence of Cre and 機科activates gene expression upon Cre-煙日dependent inversion of the coding務票 sequence.

The FLEX Cre-On 身東switch consists o計草f two pairs of heterotypic Lo件算xP-variant recombinati國區on sites, namely LoxP, having the w關吃ild-type sequence, and Lox司房2272, having a mutate煙做d sequence, fla都在nking an ORF which is in the reverse (物綠antisense) orient街照ation relative to the promoter. Bot是用h LoxP variants are recognized by草飛 Cre, but only identical pairs of Lo化高xP sites can recombine with each oth暗物er and not with any other variant. T算會he LoxP and Lox2272 s低都ites are organized in an alternating 家員fashion, with an antiparallel orienta草鐵tion for each pair. I志慢n the absence of Cre recombinase, 信拍the ORF is not expressed 你作due to its antisense 民熱orientation relative t遠分o the promoter. I可快n the presence 錢雪of Cre, the LoxP and Lox2272 sites行慢 undergo recombination w間事ith the other LoxP and Lox2272 s小報ites respectively, resulting in the inv道上ersion of the ORF to a sense orient拍村ation and excision of 月嗎one from each pair of i頻美dentical recombinati來服on sites. This allows 車個the user-selecte公長d promoter to drive the tra得美nscription of the 木電gene of interest. Since the ORF is now 森明flanked by two di化微fferent LoxP-va聽看riant sites, no呢靜 further recombination events will ta多美ke place even wh計爸en Cre is present.

The Tol2 vector system 裡河is technically si很理mple, utilizing transfection (r請錯ather than viral transducti坐東on) to permanently integrate you唱知r gene(s) of interest 機鐘into the host genome.紅玩 The Tol2 syst說新em comprises two comp你也onents: the transp車嗎oson plasmid and the tran小嗎sposase. The transposon pla廠體smid contains two inv體公erted terminal repeats (ITRs這對) bracketing the 服可region to be transp購會osed. The FLEX Cre-On switch describ路短ed above is clo頻微ned into this region. Th視文e transposase can be deliver都為ed into target cells thro明腦ugh two methods. The hel照分per plasmid can be tran現爸siently transfected into cells. A子商lternatively, target 湖見cells can be injected wit在們h transposase mRNA generated 遠火by in vitro transcription 算山from the helper plasmid. When th少光e transposon and help銀快er plasmids are 畫拍co-transfected into target 又林cells, the transposase 和和produced from the helper plasmid wo都林uld recognize the two ITRs on th遠妹e transposon and insert th個湖e flanked region including th城土e two ITRs into the host genome. 爸還At each insertion site, the T報笑ol2 transposase c家技reates an 8 bp d紙雨uplication, resulting in化司 identical 8 bp direct repeats flankin河看g each transposon在樹 integration site in the genome. I還我nsertion occurs wi和習thout any significant bias for the i我藍nsertion site se家新quence. This is unlike 她花transposon systems which have sp唱銀ecific target consensus微長 sites. For exam光服ple, piggyBac transposons 說什are typically insert明紙ed at sites conta腦店ining the sequence TTAA. Gene expressi草物on can then be activated in錢舞 the presence of Cre upon Cre-mediated 習請inversion of the 做生coding sequence. Through both methods這日 of delivering transposase, i問歌t is expressed for only a s還費hort time. Upon the l湖暗oss of the helper plasmid 技區or degradation of transposase mRNA, 姐喝the integration of the trans唱笑poson into the host ge商書nome becomes permanent.

Tol2 is a class II 們的transposon, meaning that志舊 it moves in a cut-and-paste manner, ho光請pping from place to place wi微筆thout leaving copies behind. (問文In contrast, class I transpos很知ons move in a copy-and-paste ma舞舞nner.) If Tol2 t海放ransposase is rein理技troduced into the cells, the這新 transposon could get excised件照 from the genome 章外of some cells, resulting in ei師購ther precise or imprecise exci和西sions with inde業煙ls created.

While this vecto那體r system can be used in你玩 tissue culture cells, it is part又我icularly suitable光照 for the generation o內廠f transgenic animals. When畫我 a transgenic animal carrying such a v藍大ector system is crossed to 謝吧an animal carrying a tissue-specific Cr市通e transgene, th拿作e gene of intere可匠st would be turned on妹些 in the progeny anim畫化als carrying both types車不 of transgenes, specifically in cell場明s where the tissue-specific 雨討Cre is expressed and the user-se體門lected promoter driving the gene o小輛f interest is active.

For further information abou他他t this vector system, pl有吧ease refer to the papers below.