哺乳動物shRNA表達慢病毒載體

我們的U6 shRNA慢病毒載體經(jīng)過(guò)基因敲志喝低效率驗證，結果如下圖所示。結果信湖展現了U6 shRNA與miR30 s金下hRNA之間的敲低效率比較。

圖1 U6 shRNA慢病毒載體與miR30 shRNA慢病毒載體對為微(duì)EGFP的敲低效率比較市和。(A)U6啓動子驅動的shRNA表達的慢病毒載體睡去與miR30 shRNA（4條s樂道hRNA）慢病毒載體分别包裝成(chéng)慢病毒，然後(hòu)房錯轉導穩定表達EGFP的HEK293T細胞。藥篩前後(hòu)志城流式細胞術檢測EGFP表達情況。（B）藥篩前弟坐，EGFP表達在U6 shRNA的作用下減少了46%大能（P<0.001）,在CMV啓動子驅動表達的miR30 shRNA（上不單shRNA）作用下減少了13%（P<0.001），在CMV舊件啓動子驅動表達的miR30 shRNA慢相（4條shRNA）作用下減少了44%（P<0.001）。（C）日你EGFP表達在U6 shRNA的作用下減少了72%（P<0.0動時01）,在CMV啓動子驅動表達的miR30 shRNA遠懂（單shRNA）作用下減少了60%（P&睡有lt;0.001），在CMV啓動子驅吧西動表達的miR30 shRNA（4條shR月厭NA）作用下減少了67%（P<0.001）。EGFP的相對但水(duì)表達量通過(guò)轉導與未轉導細胞的熒光強度中值（Med新事ian fluorescence intensities，MFI）的雪筆比值計算。三次重複實驗，圖中顯示SD值，p值根據Tukey檢驗計算。化機

技術複雜：使用慢病毒載體時(shí)，需要在包裝細胞中産生去土活病毒，然後(hòu)測定病毒滴度。因此慢病毒轉染相對(duì)于常規質粒轉麗業染，技術難度更高，周期更長(cháng)

永久性幹擾：慢病毒整合到宿主細胞基因組是一個不可逆的過(g體訊uò)程，U6啓動子能(néng)驅動shRNA的組成(chéng)型表達，月時對(duì)于靶基因的幹擾通常是穩定和永久的。 一旦慢病校花毒shRNA幹擾載體對(duì)基因花年産生了幹擾，目的基因的表達就(ji舊林ù)很難被(bèi)重新激活。 根據不同的實驗目的，這(線們zhè)有可能(néng)是優勢也可能(néng)是劣勢。

RSV promoter: Rous sarcoma virus promote很那r. It drives tran鄉樂scription of viral RNA in pac店黃kaging cells. This RNA is the南人n packaged into live virus.

Δ5' LTR: A deleted version of the HIV-1 5' l票的ong terminal repeat. In wildty還說pe lentivirus, 5' LTR地海 and 3' LTR are essen兒訊tially identical in sequence. The店喝y reside on two ends of the viral知文 genome and point in the same directio去到n. Upon viral integration, th見動e 3' LTR sequence is copied onto the公吧 5' LTR. The LTRs carry自輛 both promoter and p知美olyadenylation function, such that很遠 in wildtype virus, the但議 5' LTR acts as a promote答亮r to drive the transcription of the vir下為al genome, while店畫 the 3' LTR acts 兵嗎as a polyadenylation你一 signal to terminate the upstream tran文公script. On our 河白vector, Δ5' LTR is dele唱電ted for a region th知公at is required for the LTR's p船一romoter activity normally facil森要itated by the vira們明l transcription factor 樂朋Tat. This does not affect the多答 production of viral 線知RNA during pack暗了aging because the promot讀能er function is supplem他雪ented by the RSV p雨兵romoter engineered upstream of Δ歌行5' LTR.

Ψ: HIV-1 packaging signal required for th裡農e packaging of viral RNA into上友 virus.

RRE: HIV-1 Rev response element. It見水 allows the nuclear expor但章t of viral RNA b黑放y the viral Rev pro著些tein during viral pa月件ckaging.

cPPT: HIV-1 Central polypurine tract. It crea報去tes a "DNA flap" that increa舊風ses nuclear importati有去on of the viral genome during target ce話還ll infection. This improves就得 vector integration into the h國西ost genome, resulting in higher tra爸弟nsduction efficiency.在志

U6 Promoter: Drives expression of the shRNA. This科影 is the promoter of th信都e human U6 snRNA gene, an RNA polymer影內ase III promoter which ef議美ficiently express白店es short RNAs.

Sense, Antisense: These sequences are derived遠視 from your target sequences, and are tr樹慢anscribed to form t林呢he stem portion of the “hairpin” str店事ucture of the shRNA.湖但

Loop: This optimized sequenc國謝e is transcribed to風舞 form the loop por高舊tion of the shR西還NA “hairpin” stru間我cture.

Terminator: Terminates transcription of 公吧the shRNA.

hPGK promoter: Human phosphoglycerate kinas月場e 1 gene promoter. It drives the 黃吧ubiquitous expre國文ssion of the downstream marker g西要ene.

Marker: A drug selection gene (such as 得美neomycin resistance),草林 a visually detectable 睡他gene (such as EGFP), or a朋風 dual-reporter gene (such as E從熱GFP/Neo). This allows cells tra車錯nsduced with the vector to be se知現lected and/or vis線照ualized.

WPRE: Woodchuck hepatitis virus posttra的海nscriptional regulatory elem妹草ent. It enhances viral RNA stability 子為in packaging cel作慢ls, leading to 醫村higher titer of pack公慢aged virus.

ΔU3/3' LTR: A truncated version of t銀書he HIV-1 3' long terminal rep和司eat that deletes the U3 re河坐gion. This leads to the self-家你inactivation of the promoter act飛服ivity of the 5' LTR upon v理光iral vector integration into the host南錯 genome (due to the fact that匠時 3' LTR is copied onto 5' LTR d人得uring viral integration). Th議愛e polyadenylation signa家國l contained in ΔU3/3' LTR serve購就s to terminates all upstream信風 transcripts produce木煙d both during v計什iral packaging and after vir多現al integration into the農金 host genome.

SV40 early pA: Simian virus 40 early polyadenylati白公on signal. It further faci白答litates transcriptional請刀 termination after the 3' LTR 作問during viral RN畫著A transcription 學窗during packaging. This e不家levates the level of functional viral R國冷NA in packaging cells, thus improving v爸音iral titer.

Ampicillin: Ampicillin resistanc見視e gene. It allows the 資場plasmid to be maintained by amp暗行icillin selection in E. coli玩內.

pUC ori: pUC origin of replication. P做錢lasmids carrying this or短公igin exist in high 雪美copy numbers in E. co知北li.