摘要
20世纪80年代基因打靶技术的兴起极大地推动了发育生物学的研究和生物医药的开发，基因研究从最初的测序和图谱绘制逐渐发展为基因功能研究与基因致病机制解析，使现代生物学及医学研究取得了突破性进展。长期以来基因打靶技术主要在小鼠上完成，这是因为小鼠的胚胎干细胞能在体外培养并无限增殖，同时保持生殖系嵌合能力。但是利用小鼠制备的人类疾病模型往往不能真实反映人类疾病的发生和发展，甚至在诸多疾病中其症状与人类截然不同，人们一直在寻找与人的解剖和生理更为接近的大动物作为人类疾病的动物模型。
在大动物中,猪在生理结构、生化特征以及营养代谢等方面与人更为接近，被认为是目前比较理想的大动物基因修饰模型。自2000年PPL公司成功利用体细胞核移植技术制备克隆猪以来，基因修饰猪在异种器官移植研究、人类疾病模型以及农业品种改良中取得了一系列突破性成果。但是由于猪胚胎干细胞的缺乏，目前只能对体细胞进行基因打靶修饰结合核移植技术来制备基因修饰猪。而体细胞在体外培养增殖速度慢而且增殖能力有限，常规同源重组进行基因打靶的效率极低。目前，只有少量文章报道了基因修饰猪的研究。因此，基因打靶技术的研发和改进是加快基因修饰猪研究发展的关键。
近年来ZFN、TALEN以及CRISPR/Cas9技术的出现，极大的推动了基因靶向修饰技术的广泛应用，也为基因修饰猪的研究提供了更高效的工具。但在实际研究中发现，ZFN制备复杂、打靶效率低、成本高昂限制了其在基因修饰猪研究中的应用。CRISPR/Cas9虽然打靶效率高，却存在脱靶效应，不利于通过体细胞核移植制备基因修饰猪。而TALEN成本低、制备简单、打靶效率高且低脱靶效应，是目前比较理想的基因组靶向修饰工具。
TALEN是由TALE蛋白DNA结合结构域与FokI核酸酶结构域融合而成的人工核酸酶，通过单元组装法可以快速的构建针对任何靶序列的TALEN，并高效地实现对基因组特定位点的识别和切割，诱导靶序列产生双链断裂，细胞通过同源重组修复或非同源重组的末端连接实现基因靶向敲除、敲入、以及定点突变等，从而大幅度地提高基因靶向修饰的效率。目前TALEN技术已经广泛应用于真核细胞、小鼠、大鼠、斑马鱼、猪、牛等多种模式生物中，为使TALEN技术成功应用于基因修饰猪的研发，开展以下实验研究。
根据哺乳动物密码子偏好性构建哺乳动物细胞TALEN表达载体，结合已报道的Golden Gate构建体系建立适用于哺乳动物细胞的TALEN构建体系。利用DNA单链复性技术构建基于EGFP荧光蛋白的TALEN体外活性检测SSA体系，可简单、快速的对已构建TALEN的活性进行验证。利用T7内切酶可识别并切割异源二聚体DNA的特性建立了细胞内活性验证体系。为验证TALEN构建体系，选择猪pROSA26的一个靶位点设计并构建6条TALEN质粒，两两组合对TALEN在体外和细胞内的活性进行了验证。在此基础上，利用建立的TALEN体系对中国小型猪基因组进行高效且精确的定点修饰，包括基因靶向敲除、靶向敲入以及定点突变。
在基因靶向敲除实验中，针对猪DMD基因第7号外显子和猪WRN基因第3号外显子分别设计了位点特异性TALENs，转染猪胎儿成纤维细胞后经过G418筛选，成功获得猪DMD基因和WRN基因的敲除细胞株，为建立杜氏肌萎缩症和早衰症基因修饰猪模型奠定了基础；在基因靶向敲入实验中，针对猪ISL1基因设计了位点特异性TALENs，构建了基因打靶载体pFlexibleDT-ISL1-CreTd，共转染猪胎儿成纤维细胞后经过嘌呤霉素筛选，成功获得猪ISL1基因定点敲入细胞株，效率高达24%；在基因定点突变实验中，由于人胰岛素与猪胰岛素只有一个氨基酸的差别，针对猪胰岛素基因第二外显子设计了位点特异性TALENs，合成了一条具有同源性的89bp单链DNA，共转染猪胎儿成纤维细胞后经过G418筛选，成功获得将猪胰岛素B链的丙氨酸替换成苏氨酸的细胞株，并通过核移植的方式得到人源化胰岛素猪模型。
本实验首次采用TALEN技术对猪基因组内源性基因进行定点修饰，成功且高效率地实现了基因敲除、基因敲入以及定点突变等精确修饰，为大动物实现高效的基因靶向修饰提供了有力工具，也为建立各种具有重要经济价值、农业育种和医学模型的基因定点修饰猪奠定了基础。
Gene targeting technology emerged in the1980s, and has greatly advancedbiomedical research and development biology. Over the past decades, geneticresearch has developed from initial sequencing and mapping to analyzing genefunction and pathogenic mechanism. This development has resulted in tremendousbreakthroughs in modern biology and medical research. Gene targeting technologyhas been conducted in mice for a long time because their embryonic stem cells canproliferate infinitely in vitro and they can form chimeric progeny. However, mousemodels cannot accurately reflect real pathological processes and diseasedevelopment when used to mimic human diseases. These models even show oppositeresults. Thus, researchers have been investigating large animals with similaranatomy and physiology to humans for use as disease models in future studies.
Large animal models, particularly pig models, have similar physiological,biochemical, metabolic, and nutrient characteristics to human beings. Therefore, pigis regarded as one of the most ideal genetically modified animal models. Since thesuccess of cloning pigs using somatic cell nuclear transfer by PPL Company in2000,many breakthroughs have been achieved by genetically modified pigs inxenotransplantation research, human disease models, and agricultural breedimprovement. However, researchers mainly generate genetically modified pigsthrough gene targeting and nuclear transfer of somatic cells because of the lack ofporcine embryonic stem cells. Somatic cells have limited reproducibility in vitro,and conventional gene targeting using homologous recombination is inefficient.Thus, few gene targeting pig models have been reported. Improving gene targetingtechnology is the key to develop research on genetically modified pigs.
Gene targeting technology, such as zinc-finger nucleases (ZFNs), transcriptionactivator-like effector nucleases (TALENs), and clustered-regularly interspaced shortpalindromic repeat (CRISPR)/CRISPR-associated (CRISPR/Cas9) system, has obtainedsignificant breakthroughs in gene targeting. Thus, efficient tools have been developed forgene targeting research in pigs. However, ZFN is inefficient and expensive, which hinder itsapplication in pig gene targeting research. CRISPR/Cas9produces severe off-targetingeffects, which limit its applications in somatic cell targeting and nuclear transfer. Therefore,TALEN is an ideal gene targeting technology because of its high targeting efficiency, lowprice, and absence of off-target effects.
TALEN is an artificial nuclease that is fused by the binding domain of theTALE protein and nuclease domain of the Fokl protein. TALENs targeting any DNAsequence can be constructed using unit assembly. TALENs recognize and cut thespecific gene locus, and induce the double-strand break of the targeting DNAsequence. Thus, we can achieve gene knockout, knock-in, or specific point mutationusing homologous recombination repair or end connection of non-homologousrecombination. Gene targeting modification can be efficiently improved. TALEN hasbeen widely used in eukaryotic cells, mice, rats, zebrafish, pigs, cattles, and otheranimal models. TALEN is a simple method with high targeting efficiency and nooff-target effects. This study aimed to use TALEN in genetically modified pigs.
In this study, we first constructed TALEN vectors based on the preference ofmammalian codon, and built TALEN systems based on the reported Golden GateTALEN construction suitable for mammals. We developed a single-strand annealing(SSA) system using EGFP fluorescent protein and DNA single-strand renaturationtechnology. This system can validate easily and rapidly the activity of theconstructed TALENs. Simultaneously, we generated an in vivo activity detectionsystem using a T7endonuclease, which can recognize and cleave a DNAheterodimer. To validate the TALEN system established in this study, six TALENtargets targeting the pROSA26locus were designed and tested using an SSA assayand T7endonuclease I assay. Results show that the targeting efficiency mediated by TALENs in mammalian cells significantly improved. We applied this targetingmethod, including gene knockout, gene knock-in, and point mutation, to modifyefficiently and accurately the genome of Chinese mini pig.
In gene knockout, we designed TALENs targeting the seventh exon of theporcine DMD gene and third exon of the porcine WRN gene. After transfection intoporcine fetal fibroblasts with G418selection, we successfully obtained DMDknockout cells and WRN knockout cells. These cells lay the foundation to establishthe Duchenne and progeria gene-modified porcine models in the future. In geneknock-in, we designed TALENs targeting the porcine ISL1gene, and alsoconstructed the gene-targeting vector pFlexibleDT-ISL1-CreTd. Afterco-transfection into porcine fetal fibroblasts with puro selection, we obtainedISL1-targeted cells with high efficiency of up to24%. In point mutation, given thatporcine insulin has only one amino acid different from human insulin, we designedTALENs targeting the A30position of the β-chain in the porcine insulin gene. Wealso synthesized a single-stranded DNA with89bp. After transfection into porcinefetal fibroblasts with G418selection, we obtained targeted cells, in which the alaninewas successfully replaced with threonine, and humanized porcine insulin modelswere generated using the somatic cell nuclear transfer approach.
This study is the first to perform TALEN-targeting technology for the precisemodification of the porcine endogenous gene. We successfully and efficientlyachieved gene knockout, knock-in, and point mutation. The achievement of thisapproach could provide a platform for generating gene-modified porcine modelswith high efficiency. This approach could also contribute in generatinggene-modified porcine models with high economic value, improved agriculturalbreeding, and important medical applications.
引文
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