增強子/啓動子文庫構建

技術詳情

增強子/啓動子文庫的構建策略

芯片合成(chéng)寡核苷酸

芯片合成(chéng)寡核苷酸的方式非常适合用于生成(chéng)的具門亮有各種(zhǒng)大小和序列的預設計增強子/啓動上麗子變體。我們基于芯片的 DNA朋視 合成(chéng)長(cháng)度通常可達300 nt見雜，且錯誤率較低。

從基因組DNA取得

增強子/突變文庫的可變區序列可以使用探針來身雜交法從基因組DNA或細菌人工染色體（BAC）中取得問歌。這(zhè)些探針是預設計或者商業化的，專門用于短你從基因組序列或BAC中捕獲增強子或啓動子上的推著新定的調控元件。随後(hòu)，這(zhè)些區域被(bèi)克隆到載很謝體骨架中，并進(jìn)行篩選和腦化進(jìn)一步的鑒定。

基因組序列的DNA洗牌

增強子/啓動子文庫可通過(guò)D下討NA洗牌的方法構建。這(zhè)項技動作術首先將(jiāng)基因組DNA通過(guò)超聲法或酶切法破碎成(ché跳到ng)片段，然後(hòu)使用無引物的PCR或連接法并根據部分序列同源性進(討去jìn)行重組，從而將(jiāng)它們重新組裝成(電問chéng)新的嵌合序列。這(zhè)種(zh玩些ǒng)無偏向(xiàng)性的技術可以産生高度多匠計樣(yàng)性的文庫，爲了解基因組序列的自身多樣(yàng)性和調控序列優化著相提供了十分有價值的方法。

從現有調控元件定向(xiàng)進(jì高土n)化取得

增強子/啓動子文庫可以基于現有調控元件的序列又草構建。新的變體可通過(guò)各種(z從就hǒng)突變策略構建，如易錯她文PCR、簡并密碼子或DNA洗牌。這(zhè)些變體随後(hò我對u)經(jīng)過(guò)篩選以唱民鑒定哪些是與需要的表征相關的，從就花而幫助用于調控元件的定向(xiàng)進(jìn)化。

關鍵元件

增強子文庫的載體設計

圖1 增強子文庫的關鍵元件

Enhancer: The enhancer sequence拿新 to be screened can雨喝 be inserted either upstream or現家 downstream of 空懂the reporter gene. When問媽 the enhancer is pos我資itioned downstr麗些eam (A) of the reporter gene and be媽自fore the poly(A) signal, it 業好is transcribed alongside th道高e reporter gene and, ther男分efore, can be measured dir錯請ectly by mRNA-seq. Wh讀鄉en positioned upstream (B)&報鐘nbsp;of the report用術er gene, a barcode sequence is 紅少commonly incorporat見務ed in the 3’ UTR region of the repo歌很rter gene and can錢廠 be transcribed wit科廠h the reporter gene. The barcode s煙睡erves as the proxy of the enhancer&nb開銀sp;and can 匠術be sequenced 歌如;by mRNA-seq.

Minimal promoter: A user-selected minimal 訊件promoter sequence is plac家道ed here. This will drive 師司transcription of the亮離 downstream reporter if an enhan在樂cer element is present to a能聽ctivate the transcri問們ption. In the absence of such enhan話土cer, the minimal promoter w下來ill be almost co志這mpletely inactive.

點此查看其它常用最小啓動子  

Kozak: Kozak consensus sequen業船ce. It is placed in front of the s美南tart codon of the ORF of interest嗎劇 because it is believed to facilitat有睡e translation in熱北itiation in euka懂金ryotes.

Reporter: A visually detectable b吧光right fluorescent protein 上城gene (such as GFP) or a ch鐵事emiluminescent protein 文術gene (such as luciferase). This a理熱llows highly sensitive detection of en街湖hancer activity.

Barcode: Enhancer library barcodes a花得re unique, short sequences within indiv來廠idual plasmids. After scr風劇eening, the read count of each barcode 些日is determined through NG街頻S analysis. By correla身內ting the unique barcode 樂鐘sequences with the specifi新她c enhancer variants, the tran光藍scriptional activity of par做制ticular enhancers can be 一畫identified.

SV40 late pA: Simian virus 40 late polyadenylation雨工 signal. It facilitates transcriptio器木nal termination of the upstream街師 ORF.

啓動子文庫的載體設計

圖2 啓動子文庫的關鍵元件

Promoter: The promoter sequ輛謝ence to be screened can是關 be inserted her嗎冷e to drive the transcrip對市tion of the reporter.

Kozak: Kozak consensus sequence. It 離著is placed in front of the start 間北codon of the ORF of interest 相藍because it is believed to faci車遠litate translation initi個費ation in eukaryotes黑服.

Reporter: A visually detectable bright fluoresc呢西ent protein gene (such as GFP) or 市吃a chemiluminescent protein gen司秒e (such as luciferase). 子站This allows highly sensitive懂長 detection of promoter activity.

Barcode: Promoter library b鐵雨arcodes are unique, short se大草quences within individual pla森為smids. After screening, 外紅the read count of each ba件中rcode is determined through NGS ana來錯lysis. By correla是為ting the unique barcode和書 sequences with the s火下pecific promoter variants, th電綠e transcriptional activity 花子of particular pro了秒moters can be identified.費報

SV40 late pA: Simian virus 40 late polyadenyl山又ation signal. It facilitates事老 transcriptional 化吧termination of the u車城pstream ORF.

實驗數據

圖3 構建針對(duì)視錐細胞的啓動子文庫并進(jìn)行驗證路海。（A）構建、篩選和驗證的工作流程。利用四現和個成(chéng)熟的感光器特異性啓動子作爲DNA洗牌的親本序列。其中一些已被美花(bèi)證明能(néng)夠驅動視錐細胞的基因表達。進(jìn)行DNA輛黃洗牌後(hòu)，將(jiāng)這(zhè雜門)些新變體序列克隆到AAV骨架中，驅動一個綠色熒光歌城蛋白（GFP）報告基因的表達。這(zhè)些重組AAV攜帶機電不同的啓動子變體被(bèi)注射到小鼠的小爸視網膜下區域。随後(hòu)，使用熒光成(chéng)像、細胞了明分選和下一代測序（NGS）篩選具有視錐細胞特異性的啓動子變體劇來。（B）定制的視錐細胞特異性啓動子文庫的驗證。爲了驗證啓動國冷子文庫的特異性，檢測了GFP的表達情況（左購兵）。觀察到的表達情況與已知的合成(chéng)的視錐細胞特異性媽國啓動子ProA1相似，後(hòu)者驅動Td Tomato的表達（中計店）。這(zhè)表明定制文庫具有針對(duì)視街高錐細胞的特異性。