引用本文
刘晓飞,
王勤,
李晓琳,
刘丹丹,
仇松寅,
林祥梅.
转基因大动物常用载体频数分析及筛查策略[J]. 畜牧兽医学报, 2017, 48(12): 2239-2248.
LIU Xiao-fei,
WANG Qin,
LI Xiao-lin,
LIU Dan-dan,
QIU Song-yin,
LIN Xiang-mei.
Frequency Analysis of Vectors Used in Genetically Modified Animals and Screening Detection Review[J]. Acta Veterinaria et Zootechnica Sinica, 2017, 48(12): 2239-2248.
转基因大动物常用载体频数分析及筛查策略
中国检验检疫科学研究院动物检疫研究所, 北京 100176
摘要：构建重组表达载体是转基因动物生产制备研究中非常关键的一步，包括构建完整的外源基因表达盒，包含目的基因、调控序列（启动子、终止子）和筛选报告基因等。本文概述了转基因大动物制备技术，归纳统计了近10年转基因猪、牛、羊制备过程中常用的载体和频数，统计结果表明，转基因猪中启动子频数从多到少依次为酪蛋白、CAG、CMV启动子，终止子频数依次为兔β-globin poly A、酪蛋白poly A、SV40 poly A和BGH poly A；转基因羊中启动子频数从多到少依次为酪蛋白、BLG和CMV启动子，终止子依次为酪蛋白poly A、BLG poly A、BGH poly A、SV40 poly A和兔β-globin poly A；转基因牛中启动子频数从多到少依次为酪蛋白、CMV、人乳清白蛋白启动子等，终止子依次为SV40 poly A、BGH poly A和酪蛋白poly A。根据统计结果提出针对启动子和终止子的多重检测和筛查策略；分析了未来转基因动物检测可能存在的问题，以期对转基因动物的监管和筛查检测方法的建立提供参考。
关键词：转基因大动物 载体 检测
Frequency Analysis of Vectors Used in Genetically Modified Animals and Screening Detection Review
The Institute of Animal Quarantine, Chinese Academy of Inspection and Quarantine, Beijing 100176, China
Abstract: The recombinant expression vector is vital in production of genetically modified animals, which includes complete exogenous gene expression cassette containing the target gene promoter, terminator and reporter gene. This paper briefly introduces the genetically modified animal production technology and counts the vector frequency commonly used in the production of genetically modified pigs, cattle and sheep during nearly 10 years. The statistical results show that promoter frequency of the genetically modified pigs from more to less are casein, CAG, CMV promoters, terminator frequency are rabbit β-globin poly A, casein poly A, SV40 poly A and BGH poly A; The promoter frequency in genetically modified sheep from more to less are casein, BLG and CMV promoters and terminator are casein poly A, BLG poly A, BGH poly A, SV40 poly A and rabbit β-globin poly A; The promoter frequency in genetically modified cattle from more to less are casein, CMV and human α-lactalbumin promoters and terminator are SV40 poly A, BGH poly A and casein poly A. Multiple detection and screening strategies aiming at the promoter and terminator are proposed according to the statistical results. This paper also analyses the possible problems in the future in genetically modified animal detection, this review will provide reference for regulatory perspective and detection of genetically modified animals.
Key words:
genetically modified animal
vector
detection
2015年11月，FDA批准转基因大西洋鲑Aquadvantage上市，成为世界上首例被批准食用的转基因动物[1]，随之而来的，关于转基因动物的研究、检测及安全性问题成为公众关注的焦点。转基因生物(Genetically modified organisms，GMO)是指通过基因工程技术改变基因组构成的生物。转基因动物是转基因生物的一种，指通过基因工程手段将外源DNA稳定整合到动物基因组中并稳定遗传给后代的一类动物[2]。在过去的三十多年，转基因动物的研究得到了飞速的发展，转基因技术的应用研究已经涉及到动物生产的诸多方面，总体上可分为3类：第一类为提高畜产品品质、动物抗病力和减轻环境污染的转基因动物，如富含ω3脂肪酸的转基因猪[3]和牛奶中含有ω3脂肪酸的转基因牛[4]，转生长激素提高生长率的三文鱼[5]，抗禽流感的转基因鸡[6]，提高羊毛生产率的转基因绵羊[7]。第二类为用于生物制药或人类疾病模型、器官移植的转基因动物，如2009年FDA批准上市的由转基因山羊生产的抗凝血酶(商品名ATryn)[8]，表达人CD46、CD59等来减轻器官移植中免疫排斥反应的转基因猪[9]。第三类为观赏动物，如能够在美国宠物商店见到的出售带荧光的斑马鱼[10]。
转基因动物的迅速发展给转基因动物及其产品的监管带来了一系列问题，其中转基因动物的检测是关系到转基因动物监管的关键环节。在DNA水平上，按照目的片段的位置，转基因生物的检测分为筛选检测、基因特异性检测、构建特异性检测和品系特异性检测，如图 1所示，其特异性依次由低到高，检测范围依次从大到小。其中，筛选检测是对插入片段中的启动子、终止子或标记基因序列等通用元件进行检测[11]，基因特异性方法是指对外源目的基因进行检测，构建特异性方法是以转化载体中完整的基因表达盒序列为目的基因的检测方法，品系特异性检测是通过扩增基因组和插入片段的连接区域即侧翼序列来鉴定GMO的方法[12]。目前转基因植物已经建立了较为完善的转基因检测体系，如何快速准确的对转基因动物进行检测将面临巨大的挑战。为了更好更快的逐步建立起转基因动物检测体系，本文将对转基因动物的制备方法进行简述，对转基因动物制备过程中常用的载体基因进行总结，以期为转基因动物筛查检测方法的建立提供参考，并对转基因动物检测中可能存在的问题进行分析。
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图 1 转基因生物检测策略及检测特异性和范围分析 Figure 1 Detection strategy and specificity analysis of GMO |
转基因动物制备的主要传统方法：显微注射法(Microinjection)[13]、体细胞核移植法(Somatic cell nuclear transfer)[14]、逆转录病毒载体法(Retrovirus mediated gene transfer)[15]、精子载体法(Sperm-mediated gene transfer)[16]、胚胎干细胞介导法(Embryonic stem cell mediated)[17]等。每种转基因技术的优缺点各有不同，成功率取决于不同物种，科学家运用这几种转基因技术成功的生产制备了多种转基因动物。然而这些传统方法由于时间长、效率低等原因在一定程度上限制了转基因动物的发展。
近年来出现了多种新的转基因技术，如锌指核酸酶(ZFN)[18]、TALEN介导的基因打靶技术[19]、CRISPR/Cas技术[20]、RNAi介导的基因沉默技术[21]，尤其是ZFN、TALEN和CRISPR/Cas这几种基因编辑技术的出现能够更加精准的对基因进行改造，使得转基因改造的目的性更强，能够实现精确地基因敲除和定点敲入，成功率更高[22-23]。
2 转基因动物常用载体基因汇总转基因动物的生产制备研究中非常关键的一步是构建含有目的基因的重组表达载体，有病毒载体和质粒载体等，载体上包括完整的外源基因表达盒，包含目的基因、调控序列(启动子、终止子等)和筛选报告基因等。本文选取制备成功的部分转基因猪、羊、牛(共42种)，对其使用的启动子、终止子和筛选报告基因分别进行了汇总，见表 1~表 3，虽然不甚全面，但希望通过对常用载体基因的归纳，为转基因动物的筛查检测和监管提供思路和参考。
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表 1 转基因猪载体基因汇总 Table 1 Vector genes of genetically modified pigs |
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表 2 转基因羊载体基因汇总表 Table 2 Vector genes of genetically modified sheep |
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表 3 转基因牛载体基因汇总 Table 3 Vector genes of genetically modified cattle |
转基因猪载体基因汇总信息见表 1和图 2。启动子中频数最多的是酪蛋白启动子，占28%；其次是CAG启动子(由CMV早期增强子和鸡β-肌动蛋白启动子组成)，占22%；人巨细胞病毒(Cytomegalovirus，CMV)启动子、鸡β-肌动蛋白(β-actin)启动子、乳清酸蛋白(Whey acid protein，WAP)基因启动子各占11%；其他占17%。终止子中频数最多的是兔β球蛋白(β-globin)终止子，占28%；其次是酪蛋白终止子，占22%；猿猴病毒40(SV40)终止子和牛生长激素(BGH)终止子分别占11%；其他占28%。
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图 2 转基因猪启动子和终止子频数统计 Figure 2 Frequency of promoters and terminators in genetically modified pigs |
转基因羊载体基因汇总信息见表 2和图 3。启动子中频数最多的是酪蛋白启动子，占50%；其次是β-乳球蛋白(β-Lactoglobulin，BLG)启动子和CMV启动子，分别占25%和19%；还有CAG启动子，占6%。终止子中频数最多的是酪蛋白终止子，占50%；其次是BLG终止子，占22%；BGH、SV40、兔β-globin及其他分别占7%。
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图 3 转基因羊启动子和终止子频数统计 Figure 3 Frequency of promoters and terminators in genetically modified sheep |
转基因牛载体基因汇总信息见表 3和图 4。启动子中频数最多的是酪蛋白启动子，占37%；其次是CMV、乳清白蛋白启动子和人延伸因子1α，各占18%；鸡β-actin启动子占9%。终止子中SV40和BGH分别占30%；酪蛋白占10%；其他乳清白蛋白终止子等占30%。
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图 4 转基因牛启动子和终止子频数统计 Figure 4 Frequency of promoters and terminators in genetically modified cattle |
在动物转基因载体的构建中一般选择新霉素(Neomycin, neo)的抗药基因作为正向筛选标记，另外加入绿色荧光蛋白(GFP)标记基因，便可实现对外源基因的跟踪。但是为了增加转基因动物的安全性，某些报告基因常会在检测转入成功后去除。所以本文主要对启动子和终止子进行了频数分析。在转基因动物载体的构建中，启动子的选择尤其重要，因为它直接影响蛋白的表达水平和组织特异性表达位点[24]。通过对转基因猪、牛、羊等常用载体基因频数的统计，可以看出，启动子中使用最多的是酪蛋白启动子，常用的是牛αs1-casein、羊β-casein启动子[25-26]和BLG启动子。因为用于乳腺生物反应器表达蛋白的转基因动物需要乳蛋白的启动子和调控区，引导外源基因在乳腺中表达。在载体中外源基因的下游通常会插入poly A信号序列，poly A能起到稳定mRNA的作用，同时有转录终止的作用，防止和下游基因产生融合mRNA[27]。统计结果可以看出，转基因动物载体中较多的是酪蛋白poly A、β-globin poly A、SV40 poly A和BGH poly A。
3 转基因动物检测分析及展望转基因的检测中，筛选检测是以插入片段中的启动子、终止子、抗性或标记基因序列为目的片段的检测方法。因此，通过总结转基因动物制备时载体常用的基因，可以根据使用频率较高的载体基因建立筛选方法，从而对含有同一类基因的转基因动物进行初筛，提高检测的效率。本文统计的转基因猪、牛、羊启动子频数从多到少有酪蛋白启动子、CMV启动子、CAG启动子、BLG启动子、鸡β-actin启动子、WAP启动子、人延伸因子1α启动子和乳清白蛋白启动子，占总体的93%；终止子频数从多到少是酪蛋白poly A、兔β-globin poly A、SV40 poly A、BGH poly A、BLG poly A，占总体的78%。这为转基因动物筛查体系的建立提供了重要的参考，如果针对这几种启动子和终止子建立筛查方法，能够使检测覆盖到78%以上的转基因猪、牛、羊。另外，部分转基因动物由于专利保护等原因未公开载体等信息，转基因动物载体信息仍在不断更新和增加，所以可以考虑建立能够不断补充新靶标的多重筛查方法，如数字PCR方法，目前，针对部分转基因动物的筛选检测方法和基因特异性PCR检测方法的建立已有报道[71]，能够对几种转基因动物进行有效检测和筛选。
基因编辑技术的出现让精准的改造基因成为可能，能够实现精确的基因敲除和定点敲入，结合传统的转基因技术，给转基因动物领域带来了新的希望。然而基因编辑技术的应用也对转基因动物的检测带来了前所未有的挑战，基因编辑技术多数是对目的基因的删除、插入、碱基突变等，可能没有载体和外源基因的引入，仅仅存在几个碱基的删除或突变甚至多个位点的同时突变，与自然突变的基因类似从而难以区分，这使得基因水平的检测更加复杂，难以满足基因编辑产品的检测[72]。所以需要根据不同类别的转基因编辑技术探索不同的、特异的、高通量的检测方法，为转基因动物的检测和监管提供有力的技术支撑。
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