Tol2 Non-Coding RNA Exp關見ression Vector

The Tol2 non-coding RNA expressi到湖on vector is a highly裡謝 effective tool for 暗體transfection-based permanent integratio務友n of non-coding RNAs into 店人the host genome of mammalian c厭水ells. Non-coding RNAs includ快體e a wide variety of short 遠雪(<30 nucleotides) and long (>動技;200 nucleotides) functional RNA m理員olecules such as m靜術icro RNAs (miRNAs), small inte舞街rfering RNAs (si的長RNAs), piwi-inter商如acting RNAs (piR中動NAs), small nuclear RNAs (snRNAs), 報友small nucleolar RNAs (sn就些oRNAs), large intergenic n答答on-coding RNAs (lincRNAs), 農分intronic long non-coding RNA件紅s (intronic lncRNAs), natur農區al antisense transcr東藍ipts (NATs), enhancer RNAs (eRNAs)飛開 and promoter-associated RNAs (PA綠土Rs), none of which ar近現e translated into proteins, however h森著ave been found to play兵弟 important roles in many cell熱城ular processes such as DNA replicatio計很n, epigenetic regulation,業工 transcriptional and post-transcript民現ional regulation and tr空船anslation regulation.

The Tol2 non-coding RNA expression 行問vector uses an&n雪南bsp;RNA polymerase II promoter遠答 to drive the expre玩會ssion of the user-selected no照海n-coding RNA gene. Th會懂is allows the use of tissue-specific, 紙腦inducible, or variable信弟-strength promoters, en家都abling a variety of experimental appl中這ications. For RNA polymerase II-mediat制聽ed transcription, the start site is t亮的ypically in the 答話3' region of the 分都promoter while the termination site is 木農within the polyA signal sequence.路花 As a result, the玩門 transcript generated from 間得this vector does not corres媽愛pond precisely to the se畫男lected non-coding RNA gene,&nb話著sp;but contains some additional sequ為物ences both upstream and d家木ownstream. 

The Tol2 system con歌厭tains two vectors, both engineered a舞就s E. coli plasmids. On間熱e vector, referred to as the h議議elper plasmid, encodes the transpos關嗎ase. The other vector, re時森ferred to as the transpos子地on plasmid, contains two inverte小現d terminal repeats (ITRs) 又高bracketing the reg我習ion to be transposed. 弟現The non-coding RNA o遠黑f interest to be delive放金red into host cells along with a use舞睡r-selected promoter is cloned into this遠市 region of the transposon p刀鐵lasmid.

When the transposon and helper plas電自mids are co-transfect腦暗ed into target cells, the t樂吃ransposase produced from 一短the helper plasmid 錢些would recognize the tw文答o ITRs on the transposon, and家分 inserts the flan媽好ked region including the two公輛 ITRs into the host genome. Inse友懂rtion occurs without any significant b小民ias with respect to insertion site 請市sequence. This is unli可黑ke transposon system老也s which have specific ta去民rget consensus 在到sites. For example, piggyBac trans個有posons typically inserts at s說空ites containing the sequenc畫睡e TTAA.

Tol2 is a class II transposon, m話器eaning that it moves in a cut-and-呢在paste manner, hopping from城水 place to place without做多 leaving copies近信 behind. (In contrast, 雜匠class I transposons move in a co音舞py-and-paste manner.) Tol2 i黃件ntegrates as a single copy through a c水海ut-and-paste mechanism.地但 At each insertion sit花中e, the Tol2 tra答鐘nsposase creates an 8 bp duplicati飛件on, resulting in ident廠裡ical 8 bp direct repeats flankin河道g each transposon integration site in 工年the genome.

For further information about this vect妹動or system, please refer to the papers b小資elow.