Adenovirus Non-C答志oding RNA Expre些西ssion Vector

The adenovirus non-coding RNA express業知ion vector is a highly effic兵西ient vehicle for adenovir費又us-mediated delivery of頻知 non-coding RNAs of int東線erest in several m下快ammalian cell types. Non-coding R白愛NAs include a wide varie關市ty of short (<30 nucleot會男ides) and long (>200 n姐樂ucleotides) functional RNA 小從molecules such a兒風s micro RNAs (miRNAs), 短跳small interfering RNAs (si行信RNAs), piwi-interacting RNAs (piRNAs),問門 small nuclear RNAs (snRNAs),亮現 small nucleolar RNAs (snoRNAs),大分 large intergenic non-coding 明唱RNAs (lincRNAs), intronic long們什 non-coding RNAs (intronic lncRN志外As), natural antisense transcript爸訊s (NATs), enhancer RNAs (eR店務NAs) and promoter-聽中associated RNAs (PARs), n公少one of which are trans玩分lated into proteins, however have b朋秒een found to play important roles i車醫n many cellular proce和好sses such as DNA replication, epig答會enetic regulation, transcriptional and 年他post-transcriptional regu件高lation and tran化謝slation regulat錢商ion.

The adenovirus non-coding通謝 RNA expression vector議區 uses an RNA pol務一ymerase II promoter to drive the e站東xpression of the user-selected non-c又業oding RNA gene. This allow問人s the use of tissue-specific, ind場媽ucible, or variabl放紅e-strength promoters, enabling a variet用樂y of experimental appl林開ications. For RNA polymerase II-med子銀iated transcription, the sta木懂rt site is typica朋看lly in the 3' region of靜農 the promoter while the termination見務 site is within the polyA sig費火nal sequence. As a result, the trans我船cript generated from this vect黃票or does not correspond precisely 鐵農to the selected non-coding司子 RNA gene, but contains some additi報計onal sequences both upstrea爸著m and downstream. 

The adenovirus non-cod來鐵ing RNA expression vecto風哥r is first constructed a算雜s a plasmid in E. coli厭男. The non-coding RNA of i討到nterest along with a user算火 selected promoter 說技is cloned between the two inverted 線山terminal repeats (ITRs) during醫快 vector construction. The ve匠公ctor is then transfecte技新d into packaging cells, where the r秒員egion of the vector between t店議he ITRs is packaged into live virus水醫.

When the virus is added to target cells他北, the DNA cargo is delivered into cel花城ls where it enters the nucleu什放s and remains as episomal DNA with河這out integration into the ho上道st genome. The non-coding RNA seq間謝uence placed in-between the two ITRs金他 during vector construction is introduc內他ed into target cells along wi個風th the rest of viral genome.

By design, adenoviral vect麗少ors lack the E1A, E1B and E3 genes (del路要ta E1 + delta E3). T遠姐he first two are req得冷uired for the pro如民duction of live virus (these two gene票工s are engineere話黃d into the genome農劇 of packaging cells). As a腦事 result, virus produced from the vector多件s have the important sa船一fety feature of being replication窗問 incompetent (meaning tha喝明t they can transduce ta厭風rget cells but ca拍體nnot replicate in the林大m).

For further information about店哥 this vector system, please refer雨音 to the papers below.

The adenovirus non-coding討業 RNA expression vector 花作is derived from the說個 adenovirus serotype 5 (A喝道d5). It is optimi飛你zed for high copy number replicatio業靜n in E. coli, high-tite得請r packaging of live virus, efficient tr山河ansduction of h他議ost cells, and high-level tran制雨sgene expression.

Non-integration of報湖 vector DNA: The adenoviral genome does n還長ot integrate into懂都 the genome of tran微路sduced cells. Rather, it exists a坐水s episomal DNA, which can be地場 lost over time, especially in divid鄉他ing cells. 

Difficulty trans他銀ducing certain cell types: While our熱藍 adenoviral vectors樹錢 can transduce many慢低 different cell討電 types including n西村on-dividing cells,坐她 it is inefficient ag內慢ainst certain cell types such a們謝s endothelia, smooth muscl友微e, differentiat兒道ed airway epithelia, peripheral從家 blood cells, neurons, and hematopoiet紙見ic cells.

Strong immunogenicity: Live virus from adenoviral ve多什ctors can elicit st醫他rong immune response 自放in animals, thus limiting certa跳雪in in vivo applic公這ations.

Technical complexity: The use of adenovi刀人ral vectors requires the producti習雪on of live virus in話白 packaging cells followe什笑d by the measurement謝問 of viral titer. These procedures are是內 technically demanding an雨數d time consuming.