PiggyBac載體系統(tǒng)可以非常高效地把外源DNA插入哺乳動(dòng)物細(xì)胞的基因組，該系統(tǒng)技術(shù)簡單，可利用質(zhì)粒轉(zhuǎn)染（非病毒轉(zhuǎn)導(dǎo)）把您所感興趣的基因長期穩(wěn)定地整合到靶細(xì)胞基因組。

該系統(tǒng)來源于piggyBac轉(zhuǎn)座子，最開始在粉紋夜蛾（Trichoplusia ni） 中發(fā)現(xiàn)并分離而來?；谛蛄型葱苑治?，piggyBac轉(zhuǎn)座子是許多常見物種共有的一類轉(zhuǎn)座子。

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“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“復(fù)制—粘貼”的方式移動(dòng)）。由于輔助質(zhì)粒是通過瞬時(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“剪切—粘貼”的機(jī)制移動(dòng)。

關(guān)于piggyBac基因表達(dá)載體系統(tǒng)的更多信息，請參考以下文獻(xiàn)。

我們的piggyBac轉(zhuǎn)座子載體與輔助質(zhì)粒是經(jīng)過優(yōu)化的，可在大腸桿菌中高拷貝復(fù)制。該載體系統(tǒng)對多種類型的靶細(xì)胞均具有高效的轉(zhuǎn)導(dǎo)效率，實(shí)現(xiàn)外源基因的高水平表達(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)簡單：通過常規(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，過程更簡單。

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。

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。

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 driving your gene of interest is placed here.

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 signal. 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.