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哺乳動(dòng)物基因LSL條件性表達(dá)piggyBac載體

概述

PiggyBac質(zhì)?;虮磉_(dá)載體系統(tǒng)利用LoxP-Stop-LoxP(LSL)表達(dá)盒,在哺乳動(dòng)物細(xì)胞和動(dòng)物體內(nèi)實(shí)現(xiàn)Cre介導(dǎo)的條件性基因激活表達(dá)。LSL表達(dá)盒包含三個(gè)重復(fù)的SV40 polyA,序列兩端為L(zhǎng)oxP位點(diǎn)。用戶選擇的啟動(dòng)子位于LSL表達(dá)盒的上游,而用戶感興趣的基因位于其下游。在Cre重組酶不存在的情況下,該表達(dá)盒完全阻斷了目的基因的正常表達(dá);當(dāng)將Cre引入攜帶該載體的細(xì)胞中時(shí),目的基因上游的LSL表達(dá)盒將會(huì)被刪除,從而目的基因能夠在用戶選擇的啟動(dòng)子下被永久激活表達(dá)。

該載體不僅適用于體外細(xì)胞培養(yǎng),更適用于轉(zhuǎn)基因動(dòng)物模型的構(gòu)建。當(dāng)攜帶這種載體的轉(zhuǎn)基因動(dòng)物與攜帶組織特異性表達(dá)Cre的轉(zhuǎn)基因動(dòng)物雜交時(shí),產(chǎn)生的動(dòng)物后代將在特定的細(xì)胞中組織特異性表達(dá)Cre,從而啟動(dòng)目的基因在該特定細(xì)胞中組織特異性表達(dá)。

在細(xì)胞培養(yǎng)中使用該載體系統(tǒng)時(shí),可將抗生素或熒光標(biāo)記添加到載體中,使轉(zhuǎn)染后的細(xì)胞可被篩選或可示蹤以便于分離出已永久整合載體的細(xì)胞。

PiggyBac系統(tǒng)包含兩個(gè)組分,一個(gè)組分為PBase轉(zhuǎn)座酶(通常為表達(dá)PBase的IVT mRNA);另一個(gè)組分被稱為轉(zhuǎn)座子質(zhì)粒,包含兩個(gè)末端重復(fù)序列(TR)以及兩者之間的被轉(zhuǎn)座區(qū)域,需要被轉(zhuǎn)座到宿主基因組中的目的基因就克隆在這個(gè)區(qū)域。

當(dāng)表達(dá)PBase的IVT mRNA和轉(zhuǎn)座子質(zhì)粒共轉(zhuǎn)染靶細(xì)胞時(shí),PBase IVT mRNA產(chǎn)生的轉(zhuǎn)座酶將會(huì)識(shí)別轉(zhuǎn)座子的兩個(gè)TR元件,然后將被轉(zhuǎn)座區(qū)和兩個(gè)TR元件插入到宿主基因組中。轉(zhuǎn)座插入通常發(fā)生在包含TTAA序列的宿主染色體位點(diǎn),并在轉(zhuǎn)座子兩側(cè)出現(xiàn)TTAA重復(fù)序列。

PiggyBac屬于II類轉(zhuǎn)座子,通過(guò)“剪切—粘貼”的機(jī)制移動(dòng),從一個(gè)地方轉(zhuǎn)座到另一個(gè)地方,而不留下序列本身(恰好相反,I類轉(zhuǎn)座子是通過(guò)“復(fù)制—粘貼”的方式移動(dòng))。由于輔助質(zhì)粒是通過(guò)瞬時(shí)轉(zhuǎn)染進(jìn)入宿主細(xì)胞的,故會(huì)逐漸丟失。隨著輔助質(zhì)粒的丟失,轉(zhuǎn)座子在宿主基因組中變成了永久整合。當(dāng)這些宿主細(xì)胞再次被輔助質(zhì)粒轉(zhuǎn)染,整合的轉(zhuǎn)座子會(huì)再次通過(guò)“剪切—粘貼”的機(jī)制移動(dòng)。

關(guān)于該載體系統(tǒng)和Cre介導(dǎo)重組的更多信息,請(qǐng)參考以下文獻(xiàn)。

參考文獻(xiàn)主題
Mol Cell Biochem. 354:301 (2011)Review of piggyBac
Cell. 122:473 (2005)Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice
EMBO J. 12:2539 (1993)Transcription blocker prevent transcriptional interference
J Biol Chem. 259:1509-14 (1984)Purification and properties of the Cre recombinase protein
Genesis. 26:99-109. (2000)Review of the Cre/LoxP recombination system

亮點(diǎn)

該載體是為在哺乳動(dòng)物細(xì)胞和動(dòng)物體內(nèi)實(shí)現(xiàn)Cre介導(dǎo)的條件性基因表達(dá)而設(shè)計(jì)的。目的基因的表達(dá)最初是沉默的,但通過(guò)與Cre重組酶的共表達(dá)后,目的基因上游的3x SV40 pA將會(huì)刪除,從而目的基因能夠在用戶選擇的啟動(dòng)子下被永久激活表達(dá)。

優(yōu)勢(shì)

基因的穩(wěn)定激活:經(jīng)過(guò)Cre重組酶處理后,阻斷下游基因轉(zhuǎn)錄的3x SV40 pA將被永久刪除,隨后客戶選擇的啟動(dòng)子能夠驅(qū)動(dòng)的目的基因的正常表達(dá)

外源基因的永久整合:常規(guī)質(zhì)粒轉(zhuǎn)染只能實(shí)現(xiàn)外源基因的瞬時(shí)表達(dá),這種外源基因會(huì)隨著宿主細(xì)胞的分裂而不斷丟失,在快速分裂的細(xì)胞中顯得尤為顯著。相反,將PiggyBac轉(zhuǎn)座子載體和輔助質(zhì)粒一起轉(zhuǎn)染到哺乳動(dòng)物細(xì)胞中,由于轉(zhuǎn)座子在轉(zhuǎn)座酶的作用下,目的基因能穩(wěn)定地整合到宿主細(xì)胞的染色體中,從而實(shí)現(xiàn)轉(zhuǎn)座子載體上攜帶的目的基因在宿主細(xì)胞中永久表達(dá)。

技術(shù)簡(jiǎn)單:通過(guò)常規(guī)轉(zhuǎn)染即可把質(zhì)粒轉(zhuǎn)入細(xì)胞,相比起病毒載體需要進(jìn)行病毒包裝,過(guò)程更簡(jiǎn)單。

載體容量大:我們的轉(zhuǎn)座子載體總?cè)萘靠蛇_(dá)30 kb,其中質(zhì)粒骨架只占3 kb,有足夠大的容量可以放置客戶所感興趣的序列。

不足之處

轉(zhuǎn)染細(xì)胞類型受限:PiggyBac載體進(jìn)入細(xì)胞依賴于轉(zhuǎn)染。不同類型的細(xì)胞,其轉(zhuǎn)染效率差異非常大。非分裂細(xì)胞通常比分裂細(xì)胞更難轉(zhuǎn)染,原代細(xì)胞比永生化細(xì)胞更難轉(zhuǎn)染,一些重要的細(xì)胞類型轉(zhuǎn)染難度更大,如神經(jīng)元和胰島β細(xì)胞。另外,質(zhì)粒轉(zhuǎn)染主要局限于體外應(yīng)用,很少應(yīng)用于體內(nèi)實(shí)驗(yàn)(但可以應(yīng)用于轉(zhuǎn)基因動(dòng)物模型制備)。以上因素在一定程度上制約了PiggyBac系統(tǒng)的應(yīng)用。

載體關(guān)鍵元件

5' ITR: 5' inverted terminal repeat. When a DNA sequence is flanked by two ITRs, the piggyBac transpose can recognize them, and insert the flanked region including the two ITRs into the host genome.

Promoter: The promoter that will drive expression of your gene of interest after treatment with Cre recombinase.

LoxP: Recombination site for Cre recombinase. When Cre is present the region flanked by the two LoxP sites will be excised.

3x SV40 late pA: Repeats of the simian virus 40 late polyadenylation signal. This terminates transcription, preventing expression of the downstream gene of interest prior to excision with LoxP-flanked region with Cre.

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.

ORF: The open reading frame of your gene of interest is placed here.

rBG pA: Rabbit β-globin polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

CMV promoter: Human cytomegalovirus immediate early promoter. It drives the ubiquitous expression of the downstream marker gene.

Marker: A drug selection gene (such as neomycin resistance), a visually detectable gene (such as EGFP), or a dual-reporter gene (such as EGFP/Neo). This allows cells transduced with the vector to be selected and/or visualized.

BGH pA: Bovine growth hormone polyadenylation. It facilitates transcriptional termination of the upstream ORF.

3' ITR: 3' inverted terminal repeat.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

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