錢秋杰，車家倩，葉露鵬，鐘伯雄

浙江大學(xué)動物科學(xué)學(xué)院，杭州 310058

piggyBac轉(zhuǎn)座系統(tǒng)的功能改進及在哺乳動物中的應(yīng)用

錢秋杰，車家倩，葉露鵬，鐘伯雄

浙江大學(xué)動物科學(xué)學(xué)院，杭州 310058

piggyBac (PB)轉(zhuǎn)座系統(tǒng)具有轉(zhuǎn)座效率高、刪除精確、半隨機插入和攜帶片段較大等優(yōu)點。但是作為一種轉(zhuǎn)基因?qū)嶒灥墓ぞ?，特別是在哺乳動物個體水平的轉(zhuǎn)基因方面，還需要提高其轉(zhuǎn)基因效率，并降低外源基因隨機插入對內(nèi)源基因破壞的風(fēng)險。近年來的研究結(jié)果顯示，PB轉(zhuǎn)座系統(tǒng)得到了進一步改進：采用 PB轉(zhuǎn)座酶與DNA特異性結(jié)合蛋白融合而構(gòu)成的融合型轉(zhuǎn)座酶，表現(xiàn)出外源片段有插入到染色體靶向位點的傾向；采用突變體篩選的方法提高了PB轉(zhuǎn)座酶的活性，獲得了只具有切除活性而沒有插入活性的新型PB轉(zhuǎn)座酶；采用PB轉(zhuǎn)座系統(tǒng)與細(xì)菌人工染色體(Bacterial artificial chromosomes, BAC)載體聯(lián)合使攜帶的外源片段長度提高到了207 kb。改進后的PB轉(zhuǎn)座系統(tǒng)在基因組研究、基因治療、誘導(dǎo)多能干細(xì)胞(Induced pluripotent stem cells, iPSCs)誘導(dǎo)及其分化方面發(fā)揮了較大的作用。文章對PB轉(zhuǎn)座系統(tǒng)的最新研究進展和應(yīng)用前景進行了綜述。

piggyBac；轉(zhuǎn)基因；靶向插入；轉(zhuǎn)座

轉(zhuǎn)座子(Transposon)又稱跳躍基因，是能夠在基因組上移動的DNA片段，由美國女科學(xué)家Barbara McClintock最早在 1951年提出[1]，并因此在 1983年獲得諾貝爾醫(yī)學(xué)及生理學(xué)獎。轉(zhuǎn)座子分為DNA轉(zhuǎn)座子(DNA transposon)和逆轉(zhuǎn)錄轉(zhuǎn)座子(Retrotransposon)兩大類，前者以“剪切-粘貼”(cut-paste)的方式將 DNA片段從原始基因位點轉(zhuǎn)移到新的基因位點，后者是將RNA經(jīng)逆轉(zhuǎn)錄酶反轉(zhuǎn)錄成DNA后再插入到新的基因位點。大量的物種基因組測序結(jié)果發(fā)現(xiàn)，轉(zhuǎn)座子在生物進化過程中具有重要作用[2]。轉(zhuǎn)座子在哺乳動物中的應(yīng)用得益于由魚類改造而來的“睡美人”轉(zhuǎn)座子(Sleeping Beauty)的應(yīng)用[3]，由此掀起了哺乳動物利用轉(zhuǎn)座子系統(tǒng)的研究熱潮[4～6]。但是“睡美人”轉(zhuǎn)座子存在過量抑制效應(yīng)和攜帶片段偏小(5 kb左右)等缺陷[7,8]，使其在轉(zhuǎn)基因應(yīng)用上受到限制。

piggyBac(PB)轉(zhuǎn)座子首次在鱗翅目昆蟲粉紋夜蛾(Trichoplusia ni)細(xì)胞系TN-368中發(fā)現(xiàn)[9]。之后，在地中海果蠅(Ceratitis capitata)、黑腹果蠅(Drosophila melanogaster)、埃及伊蚊(Aedes aegypti)和家蠶(Bombyx mori L.)等昆蟲中利用PB轉(zhuǎn)座系統(tǒng)均取得了轉(zhuǎn)基因?qū)嶒灥某晒10～13]。研究證明 PB轉(zhuǎn)座子是一個廣譜的轉(zhuǎn)座系統(tǒng)，可以在多種生物中實現(xiàn)轉(zhuǎn)座，其中包括酵母(Schizosaccharomyces pombe)[14,15]、真渦蟲(Girardia tigrina)[16]、斑馬魚(Danio rerio)[17]、雞[18]、牛[19,20]、豬[21,22]和水稻[23]等等。

PB轉(zhuǎn)座子系統(tǒng)是以“剪切-粘貼”機制進行轉(zhuǎn)座，即轉(zhuǎn)座酶切割 PB轉(zhuǎn)座子兩端反向重復(fù)序列(Inverse terminator repeats, ITR)末端的TTAA序列，將轉(zhuǎn)座子從原始基因座切下來，然后插入到基因組新的 TTAA位點上。最早應(yīng)用于轉(zhuǎn)基因?qū)嶒灥?PB轉(zhuǎn)座系統(tǒng)是二元系統(tǒng)[24]，即采用外源基因替換 PB轉(zhuǎn)座子的轉(zhuǎn)座酶基因，把轉(zhuǎn)座酶基因構(gòu)建到另一個兩端沒有ITR或ITR被破壞了的表達(dá)質(zhì)粒中。近年來也有實驗應(yīng)用一元系統(tǒng)，即將轉(zhuǎn)座酶基因序列調(diào)整到同一質(zhì)粒的PB轉(zhuǎn)座子ITR序列的外側(cè)[25]。這兩種系統(tǒng)都能使轉(zhuǎn)座片段和轉(zhuǎn)座酶基因在轉(zhuǎn)座后成功分離，實現(xiàn)轉(zhuǎn)座片段插入基因組后不再轉(zhuǎn)座，達(dá)到穩(wěn)定遺傳的目的。

PB轉(zhuǎn)座系統(tǒng)具有多方面的優(yōu)勢，轉(zhuǎn)座效率高[26]是 PB轉(zhuǎn)座系統(tǒng)能夠被普遍應(yīng)用于轉(zhuǎn)基因?qū)嶒灥闹匾蛑?。能夠攜帶較大的外源DNA片段的能力是該系統(tǒng)的另一個優(yōu)點，當(dāng)轉(zhuǎn)座片段在 14 kb以內(nèi)時，轉(zhuǎn)座效率不會顯著下降[26,27]。PB轉(zhuǎn)座系統(tǒng)還具有半隨機(semi-random)插入的特點，即轉(zhuǎn)座片段具有插入到轉(zhuǎn)錄起始位點上下游附近以及基因內(nèi)含子區(qū)域處的TTAA位點的偏好性[28]，這使得PB系統(tǒng)有利于癌基因捕獲和增強子捕獲[29,30]。PB轉(zhuǎn)座系統(tǒng)在轉(zhuǎn)座片段被切除后不會在原位點留下印跡(footprint)，基因組可以實現(xiàn)切除后精確修復(fù)[31,32]，在可逆轉(zhuǎn)基因的應(yīng)用中具有重要作用。PB轉(zhuǎn)座酶可塑性高，通過與其它功能蛋白融合或改變轉(zhuǎn)座酶的功能區(qū)域，不僅能夠改變轉(zhuǎn)座酶的活性和作用方式，也可以提高外源基因轉(zhuǎn)座的靶向性[33～35]。

PB轉(zhuǎn)座系統(tǒng)在哺乳動物中同樣也具有高效轉(zhuǎn)座活性[26]以及其他特性，使得該系統(tǒng)在基因組研究、基因治療、干細(xì)胞誘導(dǎo)和誘導(dǎo)后分化等研究領(lǐng)域獲得了廣泛的應(yīng)用[33,36～44]。

1 piggyBac轉(zhuǎn)座子的轉(zhuǎn)座機制

PB轉(zhuǎn)座子5′端和3′端各有一個不對稱的末端重復(fù)結(jié)構(gòu)域(Terminal repeat domain, TRD)，即都包含13 bp和19 bp的反向末端重復(fù)序列，不同的是5′端和 3′端的間隔序列分別為 3 bp和 31 bp，中間是2374 bp的內(nèi)部結(jié)構(gòu)域(Internal domain, ID)[45,46]。PB轉(zhuǎn)座酶在轉(zhuǎn)座片段末端TTAA與GGG之間的T-G進行單鏈切割(nick)，產(chǎn)生3′-OH；通過3′-OH攻擊互補鏈的TTAA形成短暫的發(fā)卡結(jié)構(gòu)(hairpin)，將轉(zhuǎn)座片段從基因組上切割出來；形成發(fā)卡結(jié)構(gòu)的轉(zhuǎn)座片段在轉(zhuǎn)座酶的作用下，再次被單切，產(chǎn)生 TTAA的粘性末端；最后，又以3′-OH進攻靶位點的TTAA，將目的基因插入到靶位點[47]。但是將5′和3′端TRD同時都用3′端TRD或5′端TRD代替時，并不能產(chǎn)生轉(zhuǎn)座現(xiàn)象[38]，這說明轉(zhuǎn)座酶在將目的片段轉(zhuǎn)座到新的基因位點時需要左右臂相互配合。在供體質(zhì)粒轉(zhuǎn)座到受體質(zhì)粒實驗中，PB轉(zhuǎn)座臂需要的最短 ID序列是55 bp[48]，而在供體質(zhì)粒轉(zhuǎn)座到受體基因組實驗中，5′和3′端最短ID序列長度組合分別為311 bp和235 bp[49]或333 bp和179 bp[50]，暗示轉(zhuǎn)座臂與轉(zhuǎn)座酶之間的親和力會影響PB轉(zhuǎn)座系統(tǒng)的轉(zhuǎn)座效率。

酶的功能是由結(jié)構(gòu)域的結(jié)構(gòu)決定的。以轉(zhuǎn)座的方式來看，PB轉(zhuǎn)座酶應(yīng)該至少包含結(jié)合結(jié)構(gòu)域(Binding domain)、催化結(jié)構(gòu)域(Catalytic domain)和核定位信號(Nuclear localization signals)，可能還存在轉(zhuǎn)座酶之間作用的功能域。已有研究表明，PB轉(zhuǎn)座酶的核定位信號存在于轉(zhuǎn)座酶C端第551～571氨基酸之間[51]。DDE重組酶具有結(jié)合鎂離子的催化核心域 DDE結(jié)構(gòu)域，而結(jié)構(gòu)預(yù)測和突變篩選都顯示PB轉(zhuǎn)座酶的第 268、346、447個天冬氨酸(D)也具有類似DDE結(jié)構(gòu)域的功能[47,52]，該結(jié)構(gòu)域附近的其他保守氨基酸與轉(zhuǎn)座酶的切割和插入功能有關(guān)，將第372位精氨酸殘基與第375位賴氨酸殘基突變成丙氨酸后，獲得的PB轉(zhuǎn)座酶只有剪切功能而喪失了插入功能[53]。通過氨基酸序列比較發(fā)現(xiàn)，PB轉(zhuǎn)座酶存在幾個氨基酸保守性比較豐富的區(qū)域[52]，這些保守區(qū)域很可能是轉(zhuǎn)座酶的功能區(qū)域。用染色質(zhì)免疫沉淀實驗(Chromatin immunoprecipitation, ChIP)證實PB轉(zhuǎn)座酶在無轉(zhuǎn)座臂的情況下，也可以結(jié)合到TTAA序列[35]。以上研究結(jié)果表明，PB轉(zhuǎn)座酶具有多個功能結(jié)構(gòu)域，深入地理解轉(zhuǎn)座酶的這些結(jié)構(gòu)域在轉(zhuǎn)座功能上的作用，將有利于對PB轉(zhuǎn)座酶進行改造，提高轉(zhuǎn)座酶的轉(zhuǎn)座活性，改善靶向轉(zhuǎn)座的能力。

2 piggyBac轉(zhuǎn)座子轉(zhuǎn)座效率的改善

轉(zhuǎn)基因效率是影響轉(zhuǎn)座子系統(tǒng)在基因組研究、哺乳動物轉(zhuǎn)基因、基因治療等領(lǐng)域應(yīng)用的關(guān)鍵因素。病毒載體因其插入效率高而廣泛應(yīng)用于前期的哺乳動物轉(zhuǎn)基因研究中，但是因為其攜帶片段小、易引起癌變、外源基因易沉默等缺陷，使得該系統(tǒng)在應(yīng)用上受到了限制。PB轉(zhuǎn)座系統(tǒng)已經(jīng)被證明能夠在人HEK 293細(xì)胞和小鼠胚胎干細(xì)胞中，將外源基因轉(zhuǎn)座到宿主基因組內(nèi)，轉(zhuǎn)基因效率較高，轉(zhuǎn)基因陽性細(xì)胞數(shù)/轉(zhuǎn)染前細(xì)胞總數(shù)的比率約為10-4[26]。

在小鼠胚胎干細(xì)胞中，將昆蟲源PB轉(zhuǎn)座酶的基因序列密碼子優(yōu)化成小鼠偏好的密碼子，可以使轉(zhuǎn)座效率提高20倍[36,38]；在人胚胎干細(xì)胞實驗中，PB轉(zhuǎn)座酶基因經(jīng)密碼子優(yōu)化后，轉(zhuǎn)座效率提高了10倍左右，使轉(zhuǎn)基因陽性率達(dá)到5%左右，把5′端轉(zhuǎn)座臂的反向末端重復(fù)序列ITR的第53位堿基T轉(zhuǎn)換為C(T53C)，第136位的堿基C轉(zhuǎn)換為T(C136T)，不僅能夠使轉(zhuǎn)座效率進一步提高 59%，而且能夠使攜帶的轉(zhuǎn)座片段長度增加到18 kb[36]，為插入大片段外源基因提供了有效方法。對密碼子優(yōu)化后的PB轉(zhuǎn)座酶基因序列隨機突變后，在酵母中篩選到18個極度活躍的 PB轉(zhuǎn)座酶突變體，其中的 5個突變體(I30V/G165S、S103P、M282V、S509G/N570S 和N538K)被證實在小鼠胚胎干細(xì)胞中也具有相似或更高的轉(zhuǎn)座效率，并且進一步通過突變體之間的組合找到了一個7個氨基酸發(fā)生突變、轉(zhuǎn)座活性最高的突變體hyperactive piggyBac(mPB7)，其插入活性和切除活性比突變前的轉(zhuǎn)座酶分別提高了9倍和17倍[54]。將昆蟲源PB轉(zhuǎn)座酶(iPB)突變了7個氨基酸殘基的PB轉(zhuǎn)座酶(iPB7)，與密碼子優(yōu)化的PB轉(zhuǎn)座酶(mPB)相比，不僅在人肝細(xì)胞(Huh-7 cell)中的轉(zhuǎn)基因效率提高了2倍，使陽性細(xì)胞數(shù)占轉(zhuǎn)染前細(xì)胞總數(shù)的 0.22%左右，而且能夠使外源基因在小鼠肝臟內(nèi)的表達(dá)量也增加2倍[55]。在HEK293細(xì)胞中直接比較iPB、iPB7、mPB和mPB7 4個PB轉(zhuǎn)座酶發(fā)現(xiàn)，經(jīng)密碼子優(yōu)化后的 PB轉(zhuǎn)座酶表達(dá)量比不經(jīng)密碼子優(yōu)化的轉(zhuǎn)座酶高出10倍，同時也發(fā)現(xiàn)PB7轉(zhuǎn)座酶在低劑量情況下相對于突變前的 PB轉(zhuǎn)座酶轉(zhuǎn)座效率優(yōu)勢更加明顯[56]。但是，iPB7轉(zhuǎn)座酶在果蠅和埃及伊蚊中轉(zhuǎn)座效果并不令人滿意，雖然iPB7在果蠅細(xì)胞中轉(zhuǎn)基因效率達(dá)到了 3.14×10-3，相對于原始 PB轉(zhuǎn)座效率 (1.14×10-3)有所提高，但是在個體水平轉(zhuǎn)基因?qū)嶒炛?，iPB7 轉(zhuǎn)座酶導(dǎo)致80%左右的果蠅發(fā)生了不育的現(xiàn)象[57]。

PB轉(zhuǎn)座酶末端與某些外源肽段連接后的融合蛋白也能提高PB轉(zhuǎn)座系統(tǒng)的轉(zhuǎn)座效率。GFP、His tag、Myc tag和 HIV-TAT(HIV-1 transactivator of transcription protein)通過不同的組合與 PB轉(zhuǎn)座酶融合后獲得了兩個重組轉(zhuǎn)座酶TPLGMH和ThyPLGMH，在人 HEK293細(xì)胞的轉(zhuǎn)基因?qū)嶒炛修D(zhuǎn)座效率比Myc-tagged PB轉(zhuǎn)座酶分別提高了9倍和7倍，在iPS細(xì)胞實驗中也提高了 4倍；而且不同組合的重組轉(zhuǎn)座酶在不同類型細(xì)胞中效率也有不同，在HEK293、CHO和C17.2細(xì)胞中最高的轉(zhuǎn)座效率分別為1.1%、4.5%和 9.9%[58]。HIV-TAT蛋白含有一段能夠引導(dǎo)整個蛋白進入細(xì)胞核內(nèi)的信號肽[59,60]，該信號肽與PB轉(zhuǎn)座酶融合后能夠引導(dǎo)轉(zhuǎn)座酶在細(xì)胞核聚集，從而提高了轉(zhuǎn)座效率[61]。

3 piggyBac轉(zhuǎn)座子靶向插入的改造

PB轉(zhuǎn)座系統(tǒng)介導(dǎo)的外源片段轉(zhuǎn)基因只能識別基因組中TTAA四堿基序列，具有很大的隨機性，一旦轉(zhuǎn)座片段插入到內(nèi)源基因啟動子附近或者基因內(nèi)部，就可能引發(fā)基因沉默、癌基因表達(dá)等。而且PB轉(zhuǎn)座系統(tǒng)具有插入位點偏好性，插入位點分析結(jié)果顯示近一半的轉(zhuǎn)座子片段插入到已注釋的基因上面[28,62]，因此插入片段可能干擾機體本身的內(nèi)源基因表達(dá)，影響正常的生長發(fā)育。改變PB轉(zhuǎn)座系統(tǒng)插入位點的偏好性，甚至靶向插入到基因組特定位點，將有助于基因組研究，也是基因治療的必要條件。GAL4(Galactose regulated upstream promoter element)能夠?qū)Ｒ恍缘刈R別UAS(Upstream activator sequences)序列，激活下游基因表達(dá)。GAL4之所以能夠?qū)Ｒ坏刈R別UAS序列是因為GAL4上有特異識別并結(jié)合DNA序列的DNA結(jié)合結(jié)構(gòu)域(DNA binding domain, DBD)，如果利用GAL4的DBD序列與PB轉(zhuǎn)座酶融合形成融合蛋白，那么轉(zhuǎn)座酶就可以在DBD引導(dǎo)下在含 UAS序列的特異位點發(fā)生轉(zhuǎn)座。GAL4-piggyBac在埃及伊蚊細(xì)胞供體質(zhì)粒轉(zhuǎn)座到受體質(zhì)粒實驗中表明，有 67%的轉(zhuǎn)座片段插入到預(yù)先設(shè)定的UAS序列附近的TTAA位置[33]，在人HEK293細(xì)胞供體質(zhì)粒轉(zhuǎn)座到基因組實驗中，有 24%的轉(zhuǎn)座片段插入到UAS附近的區(qū)域[63]。但是，GAL4-piggyBac的靶向插入需要在基因組上有UAS序列，而一般情況下靶位點附近不存在UAS序列，GAL4-piggyBac融合蛋白依然不能滿足自主選擇插入位點的要求。

腺關(guān)聯(lián)病毒(Adeno-associated virus)的Rep蛋白能特異性結(jié)合到 Rep識別序列(Rep recognition sequences, RRSs)，單純地將該Rep蛋白與PB轉(zhuǎn)座酶融合后并不能使 PB轉(zhuǎn)座片段特異性插入到基因組RRSs序列附近。將RRSs序列預(yù)先插入到PB轉(zhuǎn)座片段上，雖然不能達(dá)到靶向插入的目的，但是在一定程度上改變了PB轉(zhuǎn)座子插入位點的偏好性[64]。

鋅指蛋白(Zinc finger protein, ZFP)和轉(zhuǎn)錄激活子樣效應(yīng)因子(Transcription activator-like effector, TALE)能夠自主設(shè)計，具有特異性地識別和結(jié)合到對應(yīng)序列的能力[65,66]，理論上 ZFP-piggyBac和TALE-piggyBac融合蛋白可以利用靶向作用將轉(zhuǎn)座酶帶到目的位點附近，實現(xiàn)目的基因靶向插入。將改造過的Checkpoint kinase-2 (細(xì)胞周期檢查點激酶2，CHK2)-ZFP與PB轉(zhuǎn)座酶融合后，和供體轉(zhuǎn)座片段一起導(dǎo)入到人HEK293細(xì)胞內(nèi)，但是轉(zhuǎn)座片段并沒有靶向插入到基因組上ZFP識別位點附近；進一步通過預(yù)先在基因組上插入一段CHK2-ZFP識別位點和富含TTAA的序列后，盡管ZFP-piggyBac轉(zhuǎn)座酶與原始 PB轉(zhuǎn)座酶轉(zhuǎn)基因效率類似，但是兩者在ZFP識別位點附近發(fā)生轉(zhuǎn)座的細(xì)胞數(shù)占陽性細(xì)胞總數(shù)分別是43.9%和22.50%，說明ZFP-piggyBac融合蛋白確實具有一定的靶向傾向[35]。TALE相對于ZFP具有更大的自主設(shè)計的優(yōu)勢，可以直接在基因組上選擇合適的位置設(shè)計結(jié)合位點。TALE-piggyBac融合轉(zhuǎn)座酶在不經(jīng)過轉(zhuǎn)基因改造的人HEK293細(xì)胞基因組實驗上轉(zhuǎn)基因效率達(dá)到了 11%左右，但是靶向插入到 TALE識別位點附近的效率只有 0.01%～0.014%[34]。這個系統(tǒng)的靶向插入效率仍然很低。從以上實驗結(jié)果看出在插入位點選擇上，融合蛋白仍然以PB轉(zhuǎn)座酶自身功能占主導(dǎo)，融合的DNA結(jié)合蛋白靶向作用較弱。從這一點可以看到，進一步認(rèn)識和改造 PB轉(zhuǎn)座酶，將 PB轉(zhuǎn)座酶的催化區(qū)域與TALE等DNA特異性結(jié)合蛋白融合，從而將PB轉(zhuǎn)座酶改造成在基因組任意位點都能靶向插入的新型轉(zhuǎn)基因工具，是PB轉(zhuǎn)座酶的一個重要的研究方向。

4 piggyBac轉(zhuǎn)座子導(dǎo)入外源大片段基因的能力改進

基因組上存在大量的調(diào)控元件，對基因表達(dá)有重要的調(diào)控作用。但是受到轉(zhuǎn)基因載體片段長度的限制，很多調(diào)控元件并不能構(gòu)建到載體上。細(xì)菌人工染色體(BAC)載體能夠攜帶300 kb大片段DNA[67]。PB轉(zhuǎn)座系統(tǒng)具有轉(zhuǎn)座大片段基因的能力，聯(lián)合利用PB轉(zhuǎn)座系統(tǒng)與BAC載體能夠進行大片段基因轉(zhuǎn)座。將PB轉(zhuǎn)座子的左右臂序列分別構(gòu)建在BAC載體的目的基因片段的兩端，構(gòu)建了轉(zhuǎn)座片段長度分別為28 kb、70 kb和100 kb的3個BAC載體，將這些BAC載體分別與mPB轉(zhuǎn)座酶或hypPB轉(zhuǎn)座酶(即高效轉(zhuǎn)座酶mPB7)共同導(dǎo)入小鼠ES細(xì)胞中，轉(zhuǎn)基因?qū)嶒灳@得成功[27]。雖然轉(zhuǎn)座效率隨著轉(zhuǎn)座片段的長度而有所下降，但即使是 100 kb的轉(zhuǎn)座片段，經(jīng)HAT(次黃嘌呤、氨基喋呤和胸腺嘧啶核苷酸)和FIAU(非阿尿苷)藥物雙篩選后，mPB轉(zhuǎn)座酶和hypPB轉(zhuǎn)座酶的轉(zhuǎn)基因陽性率分別達(dá)到 7%和29%[27]。PB轉(zhuǎn)座系統(tǒng)介導(dǎo)的 BAC載體轉(zhuǎn)基因技術(shù)在人ES細(xì)胞上也獲得成功，并且將轉(zhuǎn)座片段擴大到了207 kb[68,69]。將含207 kb轉(zhuǎn)座片段的BAC載體和PB轉(zhuǎn)座酶質(zhì)粒共同導(dǎo)入到HEK293細(xì)胞中，抗生素篩選得到 60個左右陽性細(xì)胞克隆，反向 PCR實驗證明有13個細(xì)胞克隆真正含有轉(zhuǎn)座片段[68]。利用原核顯微注射技術(shù)共同注射PB轉(zhuǎn)座酶和BAC載體到小鼠受精卵中也能獲得轉(zhuǎn)基因個體[70]。

5 piggyBac轉(zhuǎn)座子一元轉(zhuǎn)座系統(tǒng)的改進

供體質(zhì)粒和轉(zhuǎn)座酶輔助質(zhì)粒構(gòu)成的 PB二元轉(zhuǎn)座系統(tǒng)在細(xì)胞上能夠較好地行使轉(zhuǎn)座功能，但是在單精子胞漿內(nèi)注射(Intracytoplasmic sperm injection, ICSI)實驗中轉(zhuǎn)座效率很低。 一個自我失活(Self-inactivating)質(zhì)粒可以實現(xiàn)PB轉(zhuǎn)座系統(tǒng)在ICSI中的應(yīng)用[71]，該 PB轉(zhuǎn)座系統(tǒng)中轉(zhuǎn)座片段序列和轉(zhuǎn)座酶基因序列包含在同一個質(zhì)粒中，轉(zhuǎn)座酶基因的啟動子構(gòu)建在轉(zhuǎn)座片段的ITR內(nèi)，而轉(zhuǎn)座酶編碼基因構(gòu)建在轉(zhuǎn)座片段的ITR外，因此在轉(zhuǎn)座片段被切除后，轉(zhuǎn)座酶編碼基因與其啟動子被分離而失活。自我失活 PB轉(zhuǎn)座系統(tǒng)質(zhì)粒還被應(yīng)用在原核顯微注射實驗中，相對于傳統(tǒng)的原核顯微注射，轉(zhuǎn)基因效率提高了5倍[72]。但是該系統(tǒng)中轉(zhuǎn)座片段內(nèi)含有一段啟動子序列，可能因為PB轉(zhuǎn)座片段的隨機插入，造成該啟動子下游基因錯誤的表達(dá)，因而也存在一定的風(fēng)險。另一種PB一元轉(zhuǎn)座系統(tǒng)是將轉(zhuǎn)座片段和轉(zhuǎn)座酶構(gòu)建在一個質(zhì)粒中，并且將轉(zhuǎn)座片段末端不含TTAA序列的ITR側(cè)翼序列構(gòu)建在轉(zhuǎn)座酶基因兩側(cè)，該系統(tǒng)不僅具有與原始轉(zhuǎn)座酶相似的轉(zhuǎn)座效率，而且 5′端和3′端轉(zhuǎn)座臂片段分別減少至35 bp和63 bp，減小了插入到基因組上的轉(zhuǎn)座片段的長度[25]。

6 piggyBac轉(zhuǎn)座系統(tǒng)的應(yīng)用前景

6.1 piggyBac轉(zhuǎn)座系統(tǒng)在功能基因篩選上的應(yīng)用

傳統(tǒng)的正向遺傳學(xué)需要分子標(biāo)記篩選表型相關(guān)基因，需要檢測大量的分子標(biāo)記和雜交后代數(shù)目，耗費大量的財力和物力。轉(zhuǎn)座系統(tǒng)高效的轉(zhuǎn)座能力，以及本身即可作為分子標(biāo)記的特點，使其在尋找表型相關(guān)基因過程中能夠發(fā)揮很好的作用。PB轉(zhuǎn)座系統(tǒng)在遺傳學(xué)研究中的優(yōu)勢在于轉(zhuǎn)座效率高、插入位點分布范圍廣以及在轉(zhuǎn)座酶存在的情況下可以進行自發(fā)地轉(zhuǎn)座。在含有PB轉(zhuǎn)座片段的轉(zhuǎn)基因動物上，只要與含有PB轉(zhuǎn)座酶基因的異性同種動物交配，就可以改變轉(zhuǎn)座子插入位點[73]，能夠方便地進行大規(guī)模的功能基因篩選工作。目前，PB轉(zhuǎn)座系統(tǒng)已經(jīng)廣泛應(yīng)用于功能基因的篩選[74～76]。

6.2 piggyBac轉(zhuǎn)座系統(tǒng)在遺傳疾病治療上的應(yīng)用

基因治療的目標(biāo)是通過靶位點基因突變(敲除或插入)，使得靶基因喪失功能或者重新恢復(fù)正常功能。鋅指核酸酶(Zinc finger nuclease, ZFN)[57]、TALE核酸酶(Transcription activator-like effector nucleases, TALEN)[58,77]和CRISPR(Clustered regularly interspaced short palindromic repeats, 成簇規(guī)律間隔的短回文重復(fù)序列 )/Cas9(CRISPR-associated)系統(tǒng)[78,79]等技術(shù)的發(fā)展使得基因精確敲除成為了可能，TALEN和CRISPR/ Cas9系統(tǒng)介導(dǎo)的基因敲除治療遺傳疾病已經(jīng)獲得成功[80,81]，然而插入修復(fù)仍然受到插入效率的影響。PB轉(zhuǎn)座系統(tǒng)具有高效轉(zhuǎn)座、載體攜帶片段大等優(yōu)勢，為基因插入治療提供了一種新方法。在小鼠體細(xì)胞內(nèi)通過共同注射 PB轉(zhuǎn)座酶載體質(zhì)粒和含熒光素酶基因的轉(zhuǎn)座片段，能夠檢測到熒光素酶基因穩(wěn)定插入小鼠基因組并持續(xù)表達(dá)[82]，對小鼠靜脈同時注射PB轉(zhuǎn)座酶和外源基因的載體，同樣可以檢測到外源基因在肝臟內(nèi)長期表達(dá)[37]。但是，PB轉(zhuǎn)座系統(tǒng)仍然存在一些潛在的風(fēng)險，主要是插入片段的隨機性可能干擾機體正常的基因表達(dá)或沉默基因表達(dá)。而采用基因工程改造的與TALE等融合的PB轉(zhuǎn)座酶，具有攜帶外源片段靶向插入到自主設(shè)計的基因位點的傾向，消除隨機插入帶來的安全隱患，但是該系統(tǒng)靶向效率還很低，而且存在大量的脫靶現(xiàn)象[33～35]，所以很顯然有必要進一步開發(fā)TALE等DNA特異性結(jié)合蛋白與PB轉(zhuǎn)座酶融合的新型轉(zhuǎn)基因?qū)嶒炘?，提高其靶向效率，只有這樣才能更好地應(yīng)用于基因治療。

6.3 piggyBac轉(zhuǎn)座子在制備誘導(dǎo)多能干細(xì)胞(iPS cell)上的應(yīng)用

細(xì)胞治療已經(jīng)成為第三次醫(yī)學(xué)革命的主要手段，人們可以通過移植干細(xì)胞，使機體組織器官獲得重新生長發(fā)育的能力。但干細(xì)胞由于道德倫理等因素的限制難以獲取，為了避免道德倫理的壓力，科學(xué)家發(fā)現(xiàn)已分化細(xì)胞可以在c-Myc、Klf4、Oct4和Sox2 4個因子的調(diào)控下獲得誘導(dǎo)多能干細(xì)胞(iPS cell)[83]。PB轉(zhuǎn)座子能夠?qū)⑼瑫r攜帶這4個調(diào)控因子的載體插入到染色體上并進行表達(dá)，誘導(dǎo)生成 iPS細(xì)胞，這些細(xì)胞在細(xì)胞外形、基因表達(dá)模式和表觀遺傳等方面均與胚胎干細(xì)胞一致[84]。不僅如此，在干細(xì)胞分化過程中，也能夠利用PB轉(zhuǎn)座系統(tǒng)的精確刪除功能將誘導(dǎo)iPS的因子從基因組中切除[36,40,84]，實現(xiàn)細(xì)胞治療前后基因組的一致性。已有研究通過突變和篩選，獲得了只具有切除功能而無插入功能的兩個高效 PB 轉(zhuǎn) 座 酶 突 變 體 PB7(M194V/R372A/K375A)和PB7(R372A/K375A/D450N)，使得PB轉(zhuǎn)座系統(tǒng)介導(dǎo)的iPS細(xì)胞在誘導(dǎo)分化前能夠?qū)Ｒ恍郧谐T導(dǎo)因子序列而不發(fā)生誘導(dǎo)因子序列再次轉(zhuǎn)座到基因組上的問題，并且仍然保持了切除后基因組精確修復(fù)的能力[66]。突變后的PB轉(zhuǎn)座酶使得PB轉(zhuǎn)座系統(tǒng)在iPS細(xì)胞獲得及其分化中具有其他轉(zhuǎn)基因模式所不具備的獨特優(yōu)勢。

7 展 望

PB轉(zhuǎn)座系統(tǒng)雖然獲得了廣泛的應(yīng)用，但是對轉(zhuǎn)座酶結(jié)構(gòu)的認(rèn)識仍然不清楚。通過生物信息學(xué)分析、遺傳突變體篩選和晶體衍射等方式解析轉(zhuǎn)座酶的功能區(qū)域，對提高PB轉(zhuǎn)座系統(tǒng)的應(yīng)用價值具有很大幫助。在基因組編輯方面可以通過改變轉(zhuǎn)座酶插入識別位點(TTAA)而實現(xiàn)隨機插入，也可以與結(jié)合蛋白(如 TALE蛋白)融合達(dá)到基因組大片段切除和靶向插入的目的。在遺傳學(xué)方面，既可以利用PB轉(zhuǎn)座子介導(dǎo)的轉(zhuǎn)基因技術(shù)加快分子育種進程，也可以用于捕獲功能基因。在細(xì)胞治療方面，能夠利用PB轉(zhuǎn)座系統(tǒng)的優(yōu)勢將誘導(dǎo)因子插入到已分化的體細(xì)胞內(nèi)獲得iPS細(xì)胞，再通過PB轉(zhuǎn)座酶的切除功能將誘導(dǎo)因子從 iPS細(xì)胞內(nèi)切除，繼而進一步誘導(dǎo)分化而獲得目的細(xì)胞。上述這些內(nèi)容可以成為今后有關(guān)PB轉(zhuǎn)座系統(tǒng)的研究目標(biāo)。

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(責(zé)任編委: 張 博)

The improvement and application of piggyBac transposon system in mammals

Qiujie Qian, Jiaqian Che, Lupeng Ye, Boxiong Zhong

College of Animal Science, Zhejiang University, Hangzhou 310058, China

The piggyBac (PB) transposon system is a useful genomic engineering tool due to its high transposition efficiency, precise excision, semi-random insertion and large cargo capacity. But, it still needs to further improve the transgenic efficiency and reduce the risk of endogenous disruption caused by the random insertion of exogenous gene, especially in transgenic experiments of individual mammals. In recent studies, the PB transposase is fused with a DNA binding protein as a chimeric protein, which can guide the transposon to pre-designed loci. Besides, PB transposases obtained by mutagenesis have dramatically enhanced transposition activity and generated a novel function which is excision competent and integration defective. Furthermore, PB transposon system can carry large exogenous DNA fragments up to 207 kb when combining with the bacterial artificial chromosome vector. So far, these modified transposon systems have been widely applied in genome studies, gene therapy and induced pluripotent stem cells (iPS cells). In this study, we review the latest studies on piggyBac transposon system and its application prospect.

piggyBac; transgene; target insertion; transposition

2014-05-27;

2014-08-29

國家重點基礎(chǔ)研究發(fā)展計劃(973計劃)項目(編號：2012CB114601)和浙江省科技廳項目(編號：2013C32048)資助

錢秋杰，碩士研究生，專業(yè)方向：轉(zhuǎn)基因家蠶絲腺生物反應(yīng)器。E-mail: qianqiujie08@163.com

鐘伯雄，教授，博士生導(dǎo)師，研究方向：家蠶分子生物學(xué)。E-mail: bxzhong@zju.edu.cn

10.3724/SP.J.1005.2014.0965

時間: 2014-9-17 17:23:03

URL: http://www.cnki.net/kcms/detail/11.1913.R.20140917.1723.002.html