技術(shù)詳情

增強(qiáng)子/啟動子文庫的構(gòu)建策略

芯片合成寡核苷酸

芯片合成寡核苷酸的方式非常適合用于生成的具有各種大小和序列的預(yù)設(shè)計(jì)增強(qiáng)子/啟動子變體。我們基于芯片的 DNA 合成長度通常可達(dá)300 nt，且錯誤率較低。

從基因組DNA取得

增強(qiáng)子/突變文庫的可變區(qū)序列可以使用探針雜交法從基因組DNA或細(xì)菌人工染色體（BAC）中取得。這些探針是預(yù)設(shè)計(jì)或者商業(yè)化的，專門用于從基因組序列或BAC中捕獲增強(qiáng)子或啟動子上的推定的調(diào)控元件。隨后，這些區(qū)域被克隆到載體骨架中，并進(jìn)行篩選和進(jìn)一步的鑒定。

基因組序列的DNA洗牌

增強(qiáng)子/啟動子文庫可通過DNA洗牌的方法構(gòu)建。這項(xiàng)技術(shù)首先將基因組DNA通過超聲法或酶切法破碎成片段，然后使用無引物的PCR或連接法并根據(jù)部分序列同源性進(jìn)行重組，從而將它們重新組裝成新的嵌合序列。這種無偏向性的技術(shù)可以產(chǎn)生高度多樣性的文庫，為了解基因組序列的自身多樣性和調(diào)控序列優(yōu)化提供了十分有價值的方法。

從現(xiàn)有調(diào)控元件定向進(jìn)化取得

增強(qiáng)子/啟動子文庫可以基于現(xiàn)有調(diào)控元件的序列構(gòu)建。新的變體可通過各種突變策略構(gòu)建，如易錯PCR、簡并密碼子或DNA洗牌。這些變體隨后經(jīng)過篩選以鑒定哪些是與需要的表征相關(guān)的，從而幫助用于調(diào)控元件的定向進(jìn)化。

關(guān)鍵元件

增強(qiáng)子文庫的載體設(shè)計(jì)

圖1 增強(qiáng)子文庫的關(guān)鍵元件

Enhancer: The enhancer sequence to be screened can be inserted either upstream or downstream of the reporter gene. When the enhancer is positioned downstream (A) of the reporter gene and before the poly(A) signal, it is transcribed alongside the reporter gene and, therefore, can be measured directly by mRNA-seq. When positioned upstream (B) of the reporter gene, a barcode sequence is commonly incorporated in the 3’ UTR region of the reporter gene and can be transcribed with the reporter gene. The barcode serves as the proxy of the enhancer and can be sequenced by mRNA-seq.

Minimal promoter: A user-selected minimal promoter sequence is placed here. This will drive transcription of the downstream reporter if an enhancer element is present to activate the transcription. In the absence of such enhancer, the minimal promoter will be almost completely inactive.

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Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.

Reporter: A visually detectable bright fluorescent protein gene (such as GFP) or a chemiluminescent protein gene (such as luciferase). This allows highly sensitive detection of enhancer activity.

Barcode: Enhancer library barcodes are unique, short sequences within individual plasmids. After screening, the read count of each barcode is determined through NGS analysis. By correlating the unique barcode sequences with the specific enhancer variants, the transcriptional activity of particular enhancers can be identified.

SV40 late pA: Simian virus 40 late polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

啟動子文庫的載體設(shè)計(jì)

圖2 啟動子文庫的關(guān)鍵元件

Promoter: The promoter sequence to be screened can be inserted here to drive the transcription of the reporter.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.

Reporter: A visually detectable bright fluorescent protein gene (such as GFP) or a chemiluminescent protein gene (such as luciferase). This allows highly sensitive detection of promoter activity.

Barcode: Promoter library barcodes are unique, short sequences within individual plasmids. After screening, the read count of each barcode is determined through NGS analysis. By correlating the unique barcode sequences with the specific promoter variants, the transcriptional activity of particular promoters can be identified.

SV40 late pA: Simian virus 40 late polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

實(shí)驗(yàn)數(shù)據(jù)

圖3 構(gòu)建針對視錐細(xì)胞的啟動子文庫并進(jìn)行驗(yàn)證。（A）構(gòu)建、篩選和驗(yàn)證的工作流程。利用四個成熟的感光器特異性啟動子作為DNA洗牌的親本序列。其中一些已被證明能夠驅(qū)動視錐細(xì)胞的基因表達(dá)。進(jìn)行DNA洗牌后，將這些新變體序列克隆到AAV骨架中，驅(qū)動一個綠色熒光蛋白（GFP）報告基因的表達(dá)。這些重組AAV攜帶不同的啟動子變體被注射到小鼠的視網(wǎng)膜下區(qū)域。隨后，使用熒光成像、細(xì)胞分選和下一代測序（NGS）篩選具有視錐細(xì)胞特異性的啟動子變體。（B）定制的視錐細(xì)胞特異性啟動子文庫的驗(yàn)證。為了驗(yàn)證啟動子文庫的特異性，檢測了GFP的表達(dá)情況（左）。觀察到的表達(dá)情況與已知的合成的視錐細(xì)胞特異性啟動子ProA1相似，后者驅(qū)動Td Tomato的表達(dá)（中）。這表明定制文庫具有針對視錐細(xì)胞的特異性。