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)寡核苷酸的方開但式非常适合用于生成(chéng)的具有各種(zhǒng裡票)大小和序列的預設計增強子/啓動子民紅變體。我們基于芯片的 DNA 合成(c討些héng)長(cháng)度通常可達300 nt，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雜車來交法從基因組DNA或細菌人工染色體新作（BAC）中取得。這(zhè)些探針是預設計或視紅者商業化的，專門用于從基因組序列或BAC什放中捕獲增強子或啓動子上的推定的調控元費醫件。随後(hòu)，這(zhè)些區域被(bèi)克隆到載體骨架中，并進(j地好ìn)行篩選和進(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)DNA兒麗洗牌的方法構建。這(zhè)項化服技術首先將(jiāng)基因組DNA通過(市視guò)超聲法或酶切法破碎成(chéng)片段，然務從後(hòu)使用無引物的PCR或連接法并根據唱自部分序列同源性進(jìn)行重組，從而將(jiān個見g)它們重新組裝成(chéng)新的嵌合序列。這(zhè)種著黃(zhǒng)無偏向(xiàng)性的技術可以産生高度多樣(yàn但火g)性的文庫，爲了解基因組序列的自身海她多樣(yàng)性和調控序列優化提供了十分有身歌價值的方法。

從現有調控元件定向(xiàng)進(jìn)化取得

增強子/啓動子文庫可以基于現有調控元件的視討序列構建。新的變體可通過(guò)各種(zhǒng)身離突變策略構建，如易錯PCR、簡并密碼子或DNA洗身好牌。這(zhè)些變體随後(hòu)經(jīng)過(guò)篩選以鑒定哪些是愛體與需要的表征相關的，從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 seque現醫nce to be screened can be inserted eit放能her upstream or downstream of the repo弟得rter gene. When the enhancer is positio那白ned downstream (A) of the reporter gene麗也 and before the poly(A) signal, 湖廠it is transcribed alongside the麗中 reporter gene and, 土務therefore, can be meas話國ured directly by mRNA-seq花機. When positione影腦d upstream (B) of the repo劇章rter gene, a barcode sequ外慢ence is commonly incorporated in th妹明e 3’ UTR region of the reporter 器得gene and can be transcribed with t自器he reporter gene. 跳黑The barcode serves as t媽物he proxy of the enhancer an場車d can be sequenced by mRNA-開用seq.

Minimal promoter: A user-select小湖ed minimal promoter sequ木睡ence is placed here. This will drive t站玩ranscription of the downst區放ream reporter if美們 an enhancer element is present to a視媽ctivate the transcription. In th水女e absence of such enhance廠計r, the minimal promoter will 我北be almost completely inactive.

點此查看其它常用最小啓動子 妹近; 

Kozak: Kozak consensus sequ是男ence. It is placed in front of t明為he start codon of the ORF of int拿在erest because i聽醫t is believed to facilitate trans笑厭lation initiation in euka又綠ryotes.

Reporter: A visually detecta東黑ble bright fluorescen中風t protein gene (such 視文as GFP) or a chemiluminescent protein 和服gene (such as l會對uciferase). This al鐘議lows highly sensitive detection of e通愛nhancer activity.

Barcode: Enhancer library barcodes間林 are unique, short sequences within 這放individual plasmids. 鐘白After screening, the read cou暗河nt of each barcode is d可我etermined through NGS analysis.黃作 By correlating the uni我醫que barcode sequences 腦議with the specif事筆ic enhancer variants, the筆黃 transcriptional activity of partic請輛ular enhancers can be ident數裡ified.

SV40 late pA: Simian virus 40 lat美做e polyadenylation signal. It facilita國務tes transcripti不南onal termination of the upstr個拍eam 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些區ence to be screened can be inserted黃動 here to drive the transcription of 這通the reporter.

Kozak: Kozak consensus seq電通uence. It is placed in front of校好 the start codon of the ORF of inte費舞rest because it is b門我elieved to facilit照弟ate translation initiati科門on in eukaryotes.

Reporter: A visually detectable bright fluor北務escent protein gene 少這(such as GFP) or a那家 chemiluminescent 懂呢protein gene (such as luci頻個ferase). This allows highly se跳用nsitive detection of 山農promoter activity.

Barcode: Promoter librar藍車y barcodes are unique,光多 short sequences within in務嗎dividual plasmids. After screenin務服g, the read count of劇長 each barcode is deter一科mined through NGS anal醫月ysis. By correlating the志公 unique barcode sequences with t通我he specific promo木玩ter variants, the t章窗ranscriptional activity of par線科ticular promoters can be 還師identified.

SV40 late pA: Simian virus 40 late polyadenylation 說還signal. It facilitat靜離es transcriptional termination of the u西放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)文街行驗證。（A）構建、篩選和驗證的工作流程。利用四個成(c學看héng)熟的感光器特異性啓動子作爲DNA洗牌的親本序冷視列。其中一些已被(bèi)證明能(néng)夠驅動視錐細胞的基因表玩輛達。進(jìn)行DNA洗牌後(hòu弟離)，將(jiāng)這(zhè)些新變體序列克隆到拍動AAV骨架中，驅動一個綠色熒光蛋白（GFP）報告我姐基因的表達。這(zhè)些重組AAV攜帶不同的啓動子變體被(bèi)注射到學紙小鼠的視網膜下區域。随後(hòu)，使用熒光嗎志成(chéng)像、細胞分選和下一代測序（NGS）篩選具有視錐細文低胞特異性的啓動子變體。（B）定制的視錐細胞特異性啓動子文庫的驗證要少。爲了驗證啓動子文庫的特異性，檢測了GFP的表達情況（左）。觀察到的表達情況現什與已知的合成(chéng)的視錐細胞特異性啓動子ProA照外1相似，後(hòu)者驅動Td To得月mato的表達（中）。這(zhè)表明定制文庫具有針對(duì)視錐細胞些知的特異性。