Regular Plasmid miR30-Ba舞電sed shRNA Knockdown Vecto報木r

Our regular plasmid miR30得讀-based shRNA kn聽和ockdown vector incor筆嗎porates an optimized micro-RNA 購下system for現體 transient knockdown of target gen到外e(s) and is 光關optimized for high copy 舞動number replicatio說技n in E. coli and high-efficiency trans現身fection. Cells transfecte吃老d with the vecto吧友r can be selected微地 and/or visualized 拍高based on marker gene expression as ch樹大osen by the user. A 師到user-selected promoter drives開年 expression of a polycistronic e技唱xpression cassette containing a us制近er-selected ORF and one or more shRNAmi廠不Rs with optimize玩還d miR30-based sequ文短ences to mediate efficient shR爸物NA processing and t件就arget gene knockdown.

Promoter choice: 筆呢Unlike standard shRNA systems, which 唱姐utilize RNA polymerase III promoters s歌生uch as U6, miR30-based shRNAs村黃 can be transcribed b金玩y diverse RNA polymerase II promoters.年用 This also enables the村醫 use of tissue-spec志很ific or inducibl長分e promoters.

Multiple shRNA co-expression:&n日草bsp;Because RNA polymerase哥就 II efficiently transcribes long RN亮喝As, multiple shR討作NAmiRs can be express刀雪ed as a polycistron 輛了from a single promoter. Therefore, this紅聽 vector is available for expressi林睡ng either single or multip還議le shRNAmiRs.  

Co-expression of a reporter ORF:&房的nbsp;A user-selected gene of inter音不est or reporter gene ORF is co遠商-expressed with the shRNAm自年iRs, as a polycistron. T湖長his facilitates答東 direct monitoring of 低男shRNA transcription.

Technical simplicity: 多街;Delivering plasmid vectors into 日明cells by conventional transfection i中明s technically straightforward, and f費村ar easier than viru拍器s-based vectors wh吃慢ich require the pa雪銀ckaging of live virus.

High-level expression到做: Conventional transfect亮到ion of plasmids can often result in 著為very high copy numbers如自 in cells (up to several thousand cop喝通ies per cell). This 了路can lead to very high exp民樂ression levels of the genes c風到arried on the vector.

Non-integration of vector DNA:&n事書bsp;Conventional transfecti事弟on of plasmid vectors is腦計 also referred to as transient transf些通ection because the匠放 vector stays mostly as epis可關omal DNA in cells without們如 integration. As a result, the 算亮knockdown of target genes achi火拍eved with the regular plasmid m說小iR30-based shRNA knockdown ve還遠ctor is typically t南化ransient, therefore making the vector唱紙 unsuitable for ap門微plications requiring long-ter算山m analysis of the kno跳都ckdown phenotype. However, plasmid資些 DNA can integrate銀廠 permanently into the 黃鐘host genome at a very low frequenc和匠y (one per 102 to 10黑訊6 cells depending on c說器ell type). If a dru場行g resistance or f就見luorescence marker is incorporate跳暗d into the plasmid這愛, cells stably inte書如grating the plasmid can be de到跳rived by drug s你舊election or cell s地歌orting after extended culture.

Limited cell type 鄉少range: The efficiency of plasmid tr又用ansfection can vary greatly from cell t不雜ype to cell type. Non-d家見ividing cells are often 門人more difficult to transfect than di草雪viding cells, and primary cells鐘科 are often harder to tra文房nsfect than immortalized cell lines弟能. Some important cell types, such as服這 neurons and pan上區creatic β cells, are notori農樹ously difficult to transfect.相制 Additionally, plasmid transfection is作小 largely limited to i長從n vitro applications刀男 and rarely used分行 in vivo.

Non-uniformity of 金門gene delivery: Although a success到廠ful transfection can靜醫 result in very hig資外h average copy number 我刀of the transfect站文ed plasmid vector per cell, this 音黃can be highly n熱醫on-uniform. Some ce刀可lls can carry many copies while others 白服carry very few or none. This is unlike 地是transduction by virus購北-based vectors which tends to r鄉家esult in relatively u分她niform gene delivery into cells.