The adenovirus 輛路miR30-based shRNA kn我但ockdown vector system is a highly間服 efficient viral vector for kn都費ocking down expres能微sion of target gene(s) in a&nb年木sp;variety of mammalian ce從東ll types. It utilizes ade草自novirus-mediated deli請能very of a polycis數子tronic expressio花討n cassette consistin東雜g of one or more miR30-based shRNAs 山北(shRNAmiR) targeting gen區煙e(s) of interest 美道and a user-select區朋ed ORF, where the 東也vector remains as ep船見isomal DNA without就藍 integration into the host g草放enome. The shRNAmiR 街書transcript is processed by endogen工醫ous, cellular micro-RNA pat答空hways to produce ma海章ture shRNAs, which facilitat遠木e degradation of target gene mRN說請As. It is the pref要做erred gene knoc唱能kdown system in vivo, often used in 房個gene therapy and vaccination.&在西nbsp;

An adenovirus miR30-ba頻煙sed shRNA vector is first constructed 光飛as a plasmid in E. col海國i. The polycistronic expressi秒短on cassette consisting of one or more 友站shRNAmiRs targeting gene(s) of i讀雪nterest and a user-selecte司學d ORF is cloned between t分飛he two inverted terminal 化弟repeats (ITRs). It is then transfecte廠是d into packaging cells, where the reg多少ion of the vector between the IT錢劇Rs is packaged into也和 live virus. When the virus is a作家dded to target cells, th輛男e DNA cargo is deli暗時vered into cells where it enters th議車e nucleus and remains as e看錯pisomal DNA without in但劇tegration into the host慢著 genome. Any gene(s) that were placed 人服in-between the two ITRs d畫計uring vector construction 問家are introduced into target睡會 cells along with the res理她t of viral genome.

Unlike conventio問水nal shRNA vectors, w湖民hich utilize RNA 房日polymerase III promoters 鄉能such as U6, miRNA-based 妹做shRNA systems a空機re placed under the control of standar來低d RNA polymerase II promoters. This al都風lows the use of tissue-specific, 和計inducible, or variable-分我strength promot著空ers, enabling a variety of experime動什ntal applicatio他數ns not possible with cons算北titutive U6 pro跳內moters.

The ability of RNA polymerase II pro要票moters to efficiently 月和transcribe long transcripts in話科 the miRNA-based 如門shRNA systems also provides白房 additional advantages relative t子訊o other knockdown vector厭東 systems. Multiple shRNAmiRs 家開can be transcri她事bed as a single polycistron鐵但, which is processed to form mature sh輛水RNAs within the cell. This allows弟嗎 knockdown of multiple genes or targe秒煙ting of multiple regio錢但ns within the same 海筆gene using a single transcr費視ipt. As a result, this vector 裡廠is available for expressing either 大睡single or multiple討內 shRNAmiRs. Secondly, in this vecto外個r system, a user-selecte友熱d protein coding gene is also po要都sitioned within the海友 same polycistron as t放木he shRNAmiRs. T月紅he expression of this ORF can be used 師從to directly monitor shRNA trans美男cription (if a 大化marker ORF is used) or can be used for用金 other purposes科裡 requiring co-ex又我pression of an ORF and s答睡hRNA(s).

By design, adenoviral vecto快音rs lack the E1A, E1B a照我nd E3 genes (delta E1 海女+ delta E3). The first two are requ房來ired for the produc船林tion of live virus 和跳(these two genes are eng小黃ineered into the genome of packagi制小ng cells). As a res一嗎ult, virus produced 湖文from the vectors have the impo行森rtant safety feature of be地司ing replication incompetent 務錢(meaning that they can transduce 樂不target cells but cannot rep土房licate in them).

For further information ab靜民out this vector system, please refer t木雜o the papers below.

Non-integration of vector DNA:&nb上農sp;The adenoviral genome does not窗廠 integrate into the genome o有會f transduced cells. Rather, it exis好計ts as episomal DNA, which can be lo光些st over time, especially in divi討店ding cells. 

Difficulty transducing certain cell 下兒types: While our adenoviral vectors 你計can transduce many differ對們ent cell types including non從吧-dividing cells, i飛男t is inefficien錯不t against certain姐車 cell types such as end好很othelia, smooth muscle, differentiate拍銀d airway epithelia, pe北拿ripheral blood cells, neu日請rons, and hematopoietic c拿亮ells.

Strong immunogenicity: Live virus fr我綠om adenoviral vectors can elicit樂厭 strong immune response in animals, t國綠hus limiting ce外學rtain in vivo applications.

Technical complexity:&n公懂bsp;The use of adenoviral vectors require雜著s the production of live virus in pac自亮kaging cells foll東子owed by the measu們亮rement of viral titer. These proc司船edures are technically 頻服demanding and time consuming.