shRNA靶點設計（shRNA t畫南arget design）

Gene knockdown

RNA interference (RNAi) has been a wi見低dely utilized meth哥的od of gene modulation for 訊說many decades. Short RNA 愛樂sequences (approximately 21-23 nuc為坐leotides) complementary to 多紙the RNA of a gene of interes個我t are introduced into target cells. 上那The exogenous RNA strand binds人動 to the complementary和不 endogenous mRNA strand. The cell森票 degrades the double-stranded RN要費A and translation does not occ什那ur, knocking down the pe樂為rformance of the gene. It is impor城笑tant to note that this是這 approach does not com森開pletely knock out the gene, as s長門ome mRNAs will not be bound and will長聽 produce functional prot門身ein.

shRNA

One common RNAi approach 事金utilizes short hairpin RNAs (也雜shRNAs). Here the笑讀 sequence is designed such林業 that a single tran白月script folds back on itsel討體f and hybridizes哥筆, forming a hairpin. Th術制is double-stranded RNA 從兒complex with its 美文internal loop is transported into th用市e cytoplasm, where it is 讀日processed by Dicer and老行 is loaded into the R唱又ISC complex. The RISC comple學厭x facilitates binding between the sile水地ncing RNA and the target mRNA, at which短雜 point the mRNA will司物 be cleaved or degra在子ded (Figure 1).

Figure 1. Production and fun問鐘ction of forms of RNAi.

Why choose shRNA?

The formation of a hairpin fro頻工m a single transcript allows for c業家ustomization in in話音troduction, duration, and regulati都電on of silencing. The plasmid codin學地g for the shRNA can be tr藍車ansfected (e.g. t機信hrough electroporation or micr服音oinjection) or tran關商sduced (using viruses such as l數廠entivirus or ad村頻enovirus). Because of the option to use麗花 viral transductio嗎喝n, shRNA vectors can b計什e tagged and/or i門上ntegrated into the geno聽動me using virus machinery and can 動開be introduced to a wid船北e range of cell types.

Designing shRNAs

VectorBuilder’s shRNA Design t街來ool allows you to input 鐵呢your sequence and receiv行公e a list of all possible shRNA sequenc錢兒es in order of knockdown score學玩. Our algorithm 小服takes each 21me服美r (every sequence of 和讀21 base pairs) and determin媽場es (1) its clonability and 數畫(2) its specificity. Clonability is inf年路luenced by the order and dist在男ribution of nucleotides. Repeats, GC c生自ontent that is too h做船igh or too low, and formation of 農錯stem loops within the shRN我亮A sequence decrease stability亮友 and therefore kno筆可ckdown score. Specificity 時錯ensures that only the target gene到好 is affected, so each candid道短ate sequence is 車兵compared to the host 作遠genome. If there are regions outsi銀兒de of the gene of interest where t服服he shRNA may bind, the knockd美通own score is decreased. High knockdown子體 scores theoretically re但關flect high performance of the東那 shRNA, but some unpredicted interacti也化ons can occur, reducing the e新呢xperimental efficienc家用y. Because there 家秒is not a guarantee that the theoretica新務l outcomes completely matc風頻h the experimental out書行comes, journals typically require c都對onfirmation of shRNA knockdown多弟 using two different shRNA業爸 sequences. To m能雜aximize the changes of hav術電ing two shRNA sequences 相車with high targeting eff們銀iciency, we offer a 3+1 service, where three shRNA sequences and on光報e control (scramble) sequence is pro這村vided.