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Article(id=1221483415413637515, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1221483414323118474, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2020-0127, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1581609600000, receivedDateStr=2020-02-14, revisedDate=1585324800000, revisedDateStr=2020-03-28, acceptedDate=null, acceptedDateStr=null, onlineDate=1769153940299, onlineDateStr=2026-01-23, pubDate=1599840000000, pubDateStr=2020-09-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1769153940299, onlineIssueDateStr=2026-01-23, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1769153940299, creator=13701087609, updateTime=1769153940299, updator=13701087609, issue=Issue{id=1221483414323118474, tenantId=1146029695717560320, journalId=1189982191388893191, year='2020', volume='55', issue='9', pageStart='1983', pageEnd='2242', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1769153940039, creator=13701087609, updateTime=1769154284415, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1221484858795279116, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1221483414323118474, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1221484858795279117, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1221483414323118474, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=2035, endPage=2042, ext={EN=ArticleExt(id=1221483415963091344, articleId=1221483415413637515, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=The Cre-loxP system and its derivative systems:methodology research and applications in neuroscience, columnId=1190335348648547107, journalTitle=Acta Pharmaceutica Sinica, columnName=Reviews, runingTitle=null, highlight=null, articleAbstract=

In scientific research, it is often needed to knock in, knock out, knock down, or overexpress a specific gene in model organisms or specific types of cells to achieve precise regulation of experimental independent variables. In this case, various transgenic mice are required. The cyclization recombinase (Cre) can directly interact with different loxP (locus X over P1) DNA sequences without any cofactors to perform specific gene knock-in or knock-out at specific targets. Because of its advantages of simple action principles, high spatial specificity, and high reorganization efficiency, the Cre-loxP system is widely used in scientific research. Furthermore, the CreERT2 system (mutant of the fusion protein of Cre and estrogen receptor ligand binding domain) and the tetracycline (Tet)-on/off system, derived from the Cre-loxP system, have made the recombination of the target gene occur in temporal-specificity on the basis of spatial-specificity. This dual specificity of time and space is indispensable for research in specific directions such as fear memory and engram cells on the basis of reducing the impacts on experimental animals. Therefore, these derived systems have broad application prospects.

, correspAuthors=Su-rong YANG, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2020 Acta Pharmaceutica Sinica. All rights reserved., copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Yang ZHANG, Lin-tao JIA, Yu-dong YAN, Ya-nan ZHAO, Yue-ming ZHANG, Su-rong YANG), CN=ArticleExt(id=1221483417380766131, articleId=1221483415413637515, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=Cre-loxP系统及其衍生系统方法学的研究和在神经科学中的应用, columnId=1190335349655180086, journalTitle=药学学报, columnName=综述, runingTitle=null, highlight=null, articleAbstract=

科研中经常需要在模式生物的特定部位或特定类型细胞内敲入、敲除、敲低或过表达特定的基因以实现对实验自变量的精确调控，这时就需要利用各种转基因小鼠。环化重组酶（cyclization recombinase，Cre）可在无任何辅助因子的作用下与不同形式的loxP（locus X over P1）DNA序列直接结合并相互作用，从而可在特定靶点进行特定基因的敲入、敲除等，具有作用原理简单、空间特异性高、重组效率高等优点而被广泛应用。在Cre-loxP系统基础上衍生出的CreERT2系统（Cre与雌激素受体配体结合域的融合蛋白突变体）以及四环素（tetracycline，Tet）-on/off系统可在空间特异性的基础上补充时间特异性，从而可以使目的基因在特定时间、特定空间发生重组。这种时间、空间的双重特异性在减少对实验动物影响的基础上对于如恐惧记忆、印迹细胞等方面的研究不可或缺，具有广阔的应用前景。

, correspAuthors=杨素荣, authorNote=null, correspAuthorsNote=
*杨素荣, Tel: 86-21-54237228, E-mail:
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A, T, C, and G represent the sequences of deoxynucleotides from the 5' end to the 3' end in the primary structure of a single loxP sequence. Purple arrows indicate cyclization recombinase (Cre) specific cleavage sites , figureFileSmall=np5r98+4gVVlxn6QEMt39w==, figureFileBig=yJqb33tmyW4sDk/TFBsYXw==, tableContent=null), ArticleFig(id=1221483421172417260, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1221483415413637515, language=EN, label=null, caption=null, figureFileSmall=YNiVHnl8iUgUg5rUP2LScA==, figureFileBig=jnYHHUcq1+e+oeR+aAzVRQ==, tableContent=null), ArticleFig(id=1221483421247914742, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1221483415413637515, language=CN, label=Figure 2, caption= Schematic diagram of CreERT2 (mutant of the fusion protein of Cre and estrogen receptor ligand binding domain) system. CreERT2 is expressed under the action of tissue-specific promoters and binds to heat shock protein 90 (HSP90) in the cytoplasm, does not enter the nucleus, and the target gene is not expressed. CreERT2 dissociates from HSP90 under the action of 4-OHT (4-hydroxytamoxifen), and enters the nucleus for gene editing. Stop: Stop sequence that prevents expression of the target gene , figureFileSmall=YNiVHnl8iUgUg5rUP2LScA==, figureFileBig=jnYHHUcq1+e+oeR+aAzVRQ==, tableContent=null), ArticleFig(id=1221483421373743867, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1221483415413637515, language=EN, label=null, caption=null, figureFileSmall=y9pnX+TwMcUA4erb6EPsHw==, figureFileBig=eJRORbNC/vWiKHpR0TQE1g==, tableContent=null), ArticleFig(id=1221483421499573002, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1221483415413637515, language=CN, label=Figure 3, caption= Schematic diagram of Tet-off system composition. The Tet-off system includes two parts, tTA (tetracycline transcription activator) and TRE (tetracycline-responsive element). Among them, tTA includes tetR (tetracycline repressor protein) region and VP16 region; TRE includes tetO7 (7 repeats of tetracycline operator DNA sequence) region, PminCMV (minimal cytomegalovirus promoter) region, and target gene region (ie, Cre). tetR region can bind to tetO7 region in the absence of doxycycline (Dox), and initiate the expression of Cre under the action of VP16 and PminCMV , figureFileSmall=y9pnX+TwMcUA4erb6EPsHw==, figureFileBig=eJRORbNC/vWiKHpR0TQE1g==, tableContent=null), ArticleFig(id=1221483421679928090, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1221483415413637515, language=EN, label=null, caption=null, figureFileSmall=X4OA3KQizRvIIF3vuUde3A==, figureFileBig=z6K6oCvMgVUL4U1QBryIfQ==, tableContent=null), ArticleFig(id=1221483421797368612, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1221483415413637515, language=CN, label=Figure 4, caption= Schematic diagram of Tet-off system. The expression of tTA under the action of a specific promoter. When Dox is present in the cells, tetR region binds to Dox but not tetO7 region, and Cre cannot be expressed. When intracellular Dox is eliminated, tTA and tetR bind to TRE tetO7. Then under the action of tTA VP16 region, the expression of Cre was started , figureFileSmall=X4OA3KQizRvIIF3vuUde3A==, figureFileBig=z6K6oCvMgVUL4U1QBryIfQ==, tableContent=null), ArticleFig(id=1221483421969335090, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1221483415413637515, language=EN, label=null, caption=null, figureFileSmall=sKDIJLepW4D5J4+Ar55dZw==, figureFileBig=JLttNTVXiHlc438IbPzung==, tableContent=null), ArticleFig(id=1221483422124524348, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1221483415413637515, language=CN, label=Figure 5, caption= Schematic diagram of Tet-on system. When there is no Dox in the cells, rteR cannot bind to tetO7, and Cre is not expressed. Only when Dox is present in the cells, rtTA, rtetR, and Dox bind to form rtTA-Dox complex can bind to TRE tetO7, and mediate the expression of Cre under the action of VP16. rtTA: Reverse tetracycline transcription activator; rtetR: Reverse tetracycline repressor protein , figureFileSmall=sKDIJLepW4D5J4+Ar55dZw==, figureFileBig=JLttNTVXiHlc438IbPzung==, tableContent=null), ArticleFig(id=1221483422241964868, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1221483415413637515, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
PromoterNeuron specificity
Vglut2 (vesicular glutamate transporter 2)Glutamatergic neurons
CamkⅡα (α subunit of Ca2+/calmodulin dependent protein kinase Ⅱ)Glutamatergic neurons
Gad1 (glutamate decarboxylase 1)GABAergic neurons
Vgat [vesicular γ-aminobutyric acid (GABA) transporter]GABAergic neurons
Th (tyrosine hydroxylase)Dopaminergic neurons
c-fosActive neurons
Arc (activity-regulated cytoskeleton-associated protein)Active neurons
Pv (parvalbumin)PV positive neurons
Vip (vasoactive intestinal polypeptide)VIP positive neurons
), ArticleFig(id=1221483422351016777, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1221483415413637515, language=CN, label=Table 1, caption=

Neuron-specific promoters

, figureFileSmall=null, figureFileBig=null, tableContent=
PromoterNeuron specificity
Vglut2 (vesicular glutamate transporter 2)Glutamatergic neurons
CamkⅡα (α subunit of Ca2+/calmodulin dependent protein kinase Ⅱ)Glutamatergic neurons
Gad1 (glutamate decarboxylase 1)GABAergic neurons
Vgat [vesicular γ-aminobutyric acid (GABA) transporter]GABAergic neurons
Th (tyrosine hydroxylase)Dopaminergic neurons
c-fosActive neurons
Arc (activity-regulated cytoskeleton-associated protein)Active neurons
Pv (parvalbumin)PV positive neurons
Vip (vasoactive intestinal polypeptide)VIP positive neurons
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Cre-loxP系统及其衍生系统方法学的研究和在神经科学中的应用
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张杨 1 , 贾林涛 1 , 闫雨冬 2 , 赵亚男 2 , 张月明 2 , 杨素荣 2, *
药学学报 | 综述 2020,55(9): 2035-2042
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药学学报 | 综述 2020, 55(9): 2035-2042
Cre-loxP系统及其衍生系统方法学的研究和在神经科学中的应用
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张杨1, 贾林涛1, 闫雨冬2, 赵亚男2, 张月明2, 杨素荣2, *
作者信息
  • 1.复旦大学基础医学院, 上海 200032
  • 2.复旦大学基础医学院药理学系, 上海 200032

通讯作者:

*杨素荣, Tel: 86-21-54237228, E-mail:
The Cre-loxP system and its derivative systems:methodology research and applications in neuroscience
Yang ZHANG1, Lin-tao JIA1, Yu-dong YAN2, Ya-nan ZHAO2, Yue-ming ZHANG2, Su-rong YANG2, *
Affiliations
  • 1. School of Basic Medical Sciences, Fudan University, Shanghai 200032, China
  • 2. Department of Pharmacology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China
出版时间: 2020-09-12 doi: 10.16438/j.0513-4870.2020-0127
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摘要
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科研中经常需要在模式生物的特定部位或特定类型细胞内敲入、敲除、敲低或过表达特定的基因以实现对实验自变量的精确调控，这时就需要利用各种转基因小鼠。环化重组酶（cyclization recombinase，Cre）可在无任何辅助因子的作用下与不同形式的loxP（locus X over P1）DNA序列直接结合并相互作用，从而可在特定靶点进行特定基因的敲入、敲除等，具有作用原理简单、空间特异性高、重组效率高等优点而被广泛应用。在Cre-loxP系统基础上衍生出的CreERT2系统（Cre与雌激素受体配体结合域的融合蛋白突变体）以及四环素（tetracycline，Tet）-on/off系统可在空间特异性的基础上补充时间特异性，从而可以使目的基因在特定时间、特定空间发生重组。这种时间、空间的双重特异性在减少对实验动物影响的基础上对于如恐惧记忆、印迹细胞等方面的研究不可或缺，具有广阔的应用前景。

环化重组酶  /  CreERT2  /  Tet-on/off  /  光遗传学  /  化学遗传学

In scientific research, it is often needed to knock in, knock out, knock down, or overexpress a specific gene in model organisms or specific types of cells to achieve precise regulation of experimental independent variables. In this case, various transgenic mice are required. The cyclization recombinase (Cre) can directly interact with different loxP (locus X over P1) DNA sequences without any cofactors to perform specific gene knock-in or knock-out at specific targets. Because of its advantages of simple action principles, high spatial specificity, and high reorganization efficiency, the Cre-loxP system is widely used in scientific research. Furthermore, the CreERT2 system (mutant of the fusion protein of Cre and estrogen receptor ligand binding domain) and the tetracycline (Tet)-on/off system, derived from the Cre-loxP system, have made the recombination of the target gene occur in temporal-specificity on the basis of spatial-specificity. This dual specificity of time and space is indispensable for research in specific directions such as fear memory and engram cells on the basis of reducing the impacts on experimental animals. Therefore, these derived systems have broad application prospects.

cyclization recombinase  /  CreERT2  /  Tet-on/off  /  optogenetics  /  chemogenetics
张杨, 贾林涛, 闫雨冬, 赵亚男, 张月明, 杨素荣. Cre-loxP系统及其衍生系统方法学的研究和在神经科学中的应用. 药学学报, 2020 , 55 (9) : 2035 -2042 . DOI: 10.16438/j.0513-4870.2020-0127
Yang ZHANG, Lin-tao JIA, Yu-dong YAN, Ya-nan ZHAO, Yue-ming ZHANG, Su-rong YANG. The Cre-loxP system and its derivative systems:methodology research and applications in neuroscience[J]. Acta Pharmaceutica Sinica, 2020 , 55 (9) : 2035 -2042 . DOI: 10.16438/j.0513-4870.2020-0127
正文
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随着神经科学的快速发展, 环化重组酶(cycliza‐ tion recombinase, Cre)-loxP (locus X over P1)系统在神经精神药物、睡眠觉醒、学习记忆和视觉产生等神经科学研究领域的应用日益广泛。本文将从Cre-loxP系统的基本组成、作用机制、衍生系统以及其在神经科学中的应用展开综述。
1 Cre-loxP系统的组成元件
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1.1 Cre
Cre是由噬菌体P1基因组中长为1 029 bp (base pair)的DNA片段编码的、由343个氨基酸组成的蛋白质, 分子质量为38.5 kD[1]。该酶由较小的氨基端结构域和较大的羧基端结构域组成, 2个结构域之间由1个短链相连, 在结合位点周围形成“夹子”结构, 能与脱氧核糖核酸(deoxyribonucleic acid, DNA)分子中的大小沟部位充分接触。在重组过程中, 氨基末端与序列的识别有关, 为保守性较弱区域; 羧基末端与DNA的结合、剪接有关系, 为强保守区[2, 3]。
Cre属于细菌和酵母重组酶的整合酶家族, 具有作用于特定DNA序列时不需要任何其他蛋白质、核苷酸等辅助因子的参与以及不额外消耗能量的特点[4]。同时, Cre的蛋白质自身序列中具有核定位信号肽, 因而在真核细胞胞质中翻译后可直接进入细胞核[5], 这一特性是许多原核细胞蛋白质所不具有的, 也是Cre得以应用在小鼠等真核生物中的重要因素。该特性也是其衍生系统构建的理论基础。
1.2 LoxP序列
LoxP序列是从大肠杆菌P1噬菌体中发现的长34 bp的DNA序列, 由2个13 bp的反向重复序列和中间8 bp的间隔序列组成, 是Cre的特异性识别结合位点。这2个loxP序列的方向和位置关系决定了其中间目标基因片段的命运(图 1)。
1.3 Cre与loxP序列的相互作用
在与DNA的相互作用中, Cre先在A、E螺旋结构的作用下形成Cre重组酶四聚体。螺旋B、D、J与DNA大沟结合; 螺旋M中包含具有切割活性的酪氨酸(tyrosine, Tyr) 324残基, 为Cre的主要活性中心; 螺旋K中的组氨酸(histidine, His) 289和精氨酸(arginine, Arg) 292残基参与重组反应[6]。当DNA片段中存在loxP序列时, Cre与DNA的亲和力大大增加, 并能特异性识别loxP序列以及完成对2个loxP序列间的DNA片段进行重组。
Cre介导2个loxP序列间的DNA片段重组是个动态且可逆的过程。根据loxP序列的位置及方向, 重组可分为3种情况[7]: ①如果2个loxP序列位于同1条DNA链上, 且方向相同, Cre能有效切除2个loxP序列间的DNA片段; ②如果2个loxP序列位于同1条DNA链上, 且方向相反, Cre能有效介导2个loxP序列间的DNA片段倒位; ③如果2个loxP序列分别位于2条DNA链或染色体上, Cre能有效介导2条DNA链的交换或染色体的易位。
2 Cre-loxP系统在小鼠体内的构建
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2.1 Cre、loxP纯合小鼠杂交
利用小鼠胚胎干细胞(embryonic stem cells, ES cells)打靶技术, 先后构建纯合的loxP及Cre小鼠, 并进行交配, 从而得到含有Cre-loxP系统的转基因小鼠。
2.1.1 构建loxP纯合小鼠
构建出结构为“-同源序列-loxP-目的基因-正筛标记基因-loxP-同源序列-负筛标记基因-”(2个loxP序列同向, 多用于基因敲除)或“-同源序列-loxP-stop-loxP-目的基因-正筛标记基因-同源序列-负筛标记基因-”(2个loxP序列同向, 多用于基因敲入、敲低和过表达)的DNA片段作为打靶载体。将该DNA片段用电穿孔、显微注射或病毒感染等方式导入ES细胞, 利用正负筛选法选出与该DNA片段同源整合的ES细胞。将筛选出的ES细胞显微注射入小鼠囊胚中, 再将注射后的囊胚植入假孕小鼠子宫内, 获得具有同源整合序列的嵌合体小鼠。将嵌合体小鼠与野生型小鼠杂交, 并用DNA印记法选出含同源整合序列的杂合子小鼠, 将杂合子小鼠杂交, 得到loxP序列纯合小鼠。
2.1.2 构建Cre纯合小鼠
以噬菌体P1基因组为模板, 设计引物, 通过聚合酶链式反应得到Cre基因片段, 构建出“-同源序列-Cre-正筛标记基因-同源序列-负筛标记基因-”的打靶载体, 利用构建loxP纯合小鼠的方法获得Cre纯合小鼠, 此时得到非条件性Cre小鼠。
如果在Cre序列前加入组织或细胞特异性的基因启动子, 使得只有表达该特异性基因的组织、细胞才能产生相应的转录因子与该启动子相互作用, 以启动基因的表达, 因此Cre只在特定的组织、细胞内表达, 此时获得条件性Cre小鼠, 即实现了Cre-loxP系统的空间特异性。表 1列举了几种常用的神经元特异性启动子。
2.1.3 杂交
将得到的纯合Cre、loxP小鼠杂交, 从而得到Cre-loxP转基因小鼠[7]。LoxP纯合小鼠与非条件性Cre小鼠杂交获得完全性转基因小鼠, 即小鼠所有细胞内均存在Cre-loxP系统; 与条件性Cre小鼠杂交获得条件性转基因小鼠, 即只有在特定的组织、细胞中存在Cre-loxP系统, 实现条件性基因编辑。
2.2 Cre小鼠与工具病毒
利用上述杂交的方法获得Cre-loxP小鼠的过程繁琐复杂, 具有实验周期长、成本高、操作难度大等弊端。随着工具病毒的发展, 越来越多的实验人员选择在条件性Cre小鼠的特定部位注射携带loxP序列和目的基因片段的病毒, 并通过构建不同序列结构的病毒, 结合RNA干扰、光遗传学、化学遗传学等技术实现多种形式的基因编辑, 如基因敲入、敲低、过表达等。
在获得条件性Cre纯合小鼠后, 构建“-loxP-stoploxP-目的基因-标记基因-”(2个loxP序列同向)或“-lox2272-loxP-目的基因反向序列-lox2272-loxP-标记基因-”[lox2272为Cre重组位点, 由loxP序列2个碱基突变而来; 2个lox2272反向, 2个loxP反向。此序列称DIO (double inverted orientation)元件] DNA片段。将DNA片段转入病毒载体中, 通常为腺相关病毒(adeno-associated virus, AAV)。在特定部位注射携带目的基因片段的AAV, 利用AAV侵染神经元、遗传物质单链DNA进入宿主细胞核和复制成双链DNA游离于细胞核中并启动转录的特性将构建的目的DNA导入注射区域的神经元内[8, 9]。Cre在组织、细胞特异性启动子的作用下可实现条件性表达, 游离于宿主细胞核中的病毒双链DNA片段在条件性Cre的作用下实现了空间特异性的基因编辑。
病毒携带的目的基因通常为化学遗传学中调控神经元活性的特定药物激活的受体(designer receptors exclusively activated by designer drugs, DREADDs)及光遗传学中调控神经元活性的光敏感离子通道蛋白。表达这些目的基因的神经元在氯氮平N-氧化物(clozapine N-oxide, CNO)或蓝光/黄光的刺激下被激活或抑制[10, 11]。病毒携带的目的基因也可以是Ca2+探针、膜电压探针和神经递质探针等荧光指示剂, 从而观察某一特定脑区或神经元的Ca2+、膜电位以及神经递质浓度的变化。
2.3 LoxP小鼠与工具病毒
与Cre小鼠联合工具病毒原理相近, 在获得loxP纯合小鼠后, 将含“-组织特异性启动子-Cre-”的DNA片段导入载体病毒中, 再将载体病毒显微注射到目标区域, 使病毒感染loxP小鼠特定部位。由于Cre在组织特异性启动子的作用下只在特定类型细胞中表达, 故可通过与小鼠loxP序列作用, 实现在特定类型细胞中进行基因调控。
2.4 Cre与loxP工具病毒
分别构建携带“-组织特异性启动子-Cre-标记基因-”与“-DIO-目的基因-标记基因-”的工具病毒AAV, 在野生型小鼠的特定部位共同注射上述2种病毒, 在特异性启动子的驱动下实现条件性基因调控。
3 Cre-loxP系统的衍生系统
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在Cre-loxP系统出现后, 科学家通过对该系统进行突变改造期望获得性能更加优越的条件性基因表达调控系统, 其中就包括可以在时间上进行特异性调控Cre发挥基因编辑功能的CreERT2系统和四环素(tetracycline, Tet)-on/off系统。CreERT2系统为翻译后水平上的调控, Tet-on/off系统是转录水平上的调控。CreERT2和Tet-on/off的主要优势体现在时间特异性上, 这与Cre-loxP系统具有的空间特异性(组织、细胞特异性)形成很好的互补作用, 在需要对目的基因表达进行时间上的调控时便会应用到衍生系统。
3.1 CreERT2系统
该系统是将Cre与人雌激素受体(estrogen receptor, ER)的配体结合区(ligand binding domain, LBD)相融合, 转录、翻译后形成定位于胞浆中的融合蛋白CreER。该蛋白不进入细胞核, 也无重组功能。只有在雌激素诱导后, 融合的Cre才通过构象变化从锚定蛋白热休克蛋白90 (heat shock protein 90, HSP90)上解离下来, 并在自身核定位信号肽的作用下进入细胞核, 识别宿主DNA上的loxP位点并发生重组。这样就可以通过控制雌激素的给药时间, 实现对基因重组时间特异性的调控。
为了避免内源性雌激素的干扰, 在CreER的ER-LBD做1个点突变(G521R)就可得到只响应外源人工合成的雌激素类似物他莫昔芬(tamoxifen)和4-羟基他莫昔芬(4-hydroxytamoxifen, 4-OHT)诱导的突变体, 命名为CreERT。而另一种CreER突变体被证明对4-OHT的敏感性远高于CreERT, 这种突变体就是CreERT2 (图 2), 其ER-LBD中存在3个点突变: C400V、M453A和L544A[12-15]。
3.2 Tet-on/off系统
该系统来源于大肠杆菌Tn10转座子上的四环素操纵子, 该操纵子主要包含四环素阻遏蛋白(tetracycline repressor protein, tetR)与四环素操纵子DNA序列(tetracycline operator DNA sequence, tetO)。当细菌内无Tet或其衍生物强力霉素(doxycycline, Dox)存在时, tetR与tetO结合, 阻止四环素抗性基因表达; 反之, 当Tet或Dox存在时, 因为Tet和Dox与tetR有极高的亲和性, 可诱使tetR蛋白的结构发生改变, 并从tetO上解离, 造成tetR对四环素操纵子的阻遏解除, 使四环素抗药蛋白表达。基于此原理, 人们设计了2种衍生的基因表达调控系统。
3.2.1 Tet-off系统(无Tet、Dox时目的基因表达)
该系统主要包括四环素转录激活因子(tetracycline transcription activator, tTA)与四环素反应元件(tetracycline- responsive element, TRE)。tTA包括tetR区和VP16区, tetR区为四环素阻遏蛋白的DNA结合区; VP16区为单纯带状疱疹病毒VP16的转录激活区域。TRE包括tetO区、PminCMV (minimal cytomegalovirus promoter)区和Cre序列。tetO区由7个头尾相连的tetO (tetO7)序列组成, 与tTA中的tetR区结合; PminCMV区为最小巨细胞病毒早期启动子, 自身启动转录的能力很低。当胞内存在Dox时, 引起tetR区变构, tTA不能与TRE结合, PminCMV区自身不足以启动Cre转录。当去除Dox时, tTA中的tetR区与TRE中的tetO区结合, 在VP16区与PminCMV区的共同作用下启动Cre的转录(图 3、4)[16-18]。
3.2.2 Tet-on系统(有Tet、Dox时目的基因表达)
该系统主要包括反义四环素转录激活因子(reverse tetracycline transcription activator, rtTA)和TRE。与Tet-off系统不同的是, rtTA包括反义tetR (reverse tetracycline repressor protein, rtetR)区和VP16区, rtetR是由tetR的4个氨基酸发生突变产生的, 突变后其产生作用的条件与tetR完全相反。只有当胞内存在Dox时, rtetR在Dox的作用下才可与tetO区结合, 启动Cre的转录, 启动下游的基因编辑。胞内无Tet/Dox时, rtetR区不能与tetO区结合, 无法启动Cre转录(图 5)[19, 20]。
4 应用举例
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本文主要举例论证Cre-loxP系统及其衍生系统在神经精神领域中的相关应用。
4.1 神经精神药物研究
氯胺酮是N-甲基-D-天冬氨酸受体(N-methyl-Daspartic acid receptor, NMDAR)阻断剂, 可快速引起内侧前额叶皮层(medial prefrontal cortex, mPFC)胞外谷氨酸水平和突触形成的增加, 但是引起这些变化和氯胺酮产生快速抗抑郁作用的机制尚不清楚。2020年耶鲁大学医学院Gerhard等[21]结合RNAi (ribonucleic acid interference)技术和Cre-loxP系统, 利用携带目的基因Glun2b shRNA (GluN2B: NMDAR的1个亚基; shRNA: short hairpin RNA)的病毒载体和CamkⅡα (α subunit of Ca2+/calmodulin dependent protein kinase Ⅱ)- Cre、Gad1 (glutamate decarboxylase 1) -Cre小鼠, 特异性地敲低了mPFC谷氨酸能神经元和GABA (γ-amino‐ butyric acid)能神经元NMDAR的Glun2b基因, 再结合强迫游泳等行为学实验, 进一步证明了mPFC中的GABA能神经元而不是谷氨酸能神经元上的NMDAR-GluN2B亚基是氯胺酮发挥快速抗抑郁作用的靶点。
4.2 睡眠觉醒
2016年斯坦福大学Eban-Rothschild等[22]使用Th(tyrosine hydroxylase) -Cre小鼠, 在中脑腹侧被盖区(ventral tegmental area, VTA)注射多种Cre依赖的病毒(携带的目的基因包括Ca2+荧光探针、光敏感离子通道和DREADDs), 特异性地记录了VTA多巴胺(dopamine, DA)能神经元在觉醒、睡眠时相的活性和选择性激活或抑制VTA DA能神经元后小鼠觉醒、睡眠时相的转换与维持时间, 进而发现VTA DA能神经元在促进小鼠觉醒中发挥重要作用。
2019年伦敦帝国理工学院的Yu等[23]使用Vglut2 (vesicular glutamate transporter 2) -Cre小鼠和Vgat (vesicular GABA transporter)-Cre小鼠, 同样结合多种Cre依赖的病毒(携带的目的基因有Ca2+荧光探针、激活、抑制性的DREADDs), 发现VTA谷氨酸能神经元和GABA能神经元分别在促进小鼠觉醒及睡眠中发挥重要作用。
近年, 作者实验室采用Cre小鼠并结合多种病毒, 分别观察到伏隔核多巴胺D1受体阳性神经元调控觉醒[24]、吻内侧被盖核神经元具有生理性睡眠促进作用[25]及视网膜神经节细胞-上丘GABA能神经元- VTA DA能神经元神经通路介导急性黑暗暴露诱发小鼠觉醒效应[26]。利用这些技术, 可观察到生理状态下某个核团某一类型神经元的激活状态, 亦可人为激活或抑制靶神经元, 从而探究该核团该类神经元在某生理过程中的关键作用。
4.3 社会行为
近年, 采用光遗传学、化学遗传学等技术, 结合Cre-loxP转基因小鼠在小鼠社会行为研究领域的应用越来越广泛。
2019年浙江大学Dong等[27]在研究丘脑网状核(thalamic reticular nucleus, TRN)相关神经环路在小鼠逃跑(flight)行为中的作用时使用Pv (parvalbumin)-Cre小鼠, 通过在TRN立体定位注射携带Ca2+荧光探针与抑制性光敏感离子通道的AAV观察, 控制PV阳性神经元的活性。研究发现, 小鼠在逃跑时TRN PV阳性神经元明显激活; 利用光遗传学技术抑制TRN PV阳性神经元时发现逃跑行为显著减少, 从而发现TRN相关神经环路在介导小鼠逃跑行为中发挥重要作用。
2019年加州大学旧金山分校Lee等[28]在研究mPFC VIP (vasoactive intestinal polypeptide)阳性神经元相关神经环路在小鼠回避行为中的作用时使用Vip- Cre小鼠, 结合携带Ca2+荧光探针与抑制性光敏感离子通道的AAV, 观察到mPFC VIP阳性神经元在发生回避行为时激活; 抑制其活性后回避行为减少, 而探索行为增加, 从而发现mPFC VIP阳性神经元在小鼠回避行为中的重要作用。
4.4 恐惧记忆
对恐惧记忆的研究有一个较为通用的范式:首先利用巴甫洛夫条件反射原理获得恐惧记忆; 其次让小鼠正常生活达到恐惧记忆的巩固; 然后提取、激活恐惧记忆, 让恐惧记忆再巩固; 最后可在再巩固的特定时间窗内再次利用巴甫洛夫条件反射原理进行恐惧记忆的消除。如果需要探究该范式不同阶段激活的神经元在恐惧记忆的消退、复发中所起的作用就需要在不同时间段内准确标记并控制神经元, 这时就需要用到兼具空间特异性与时间特异性的CreERT2或Tet-on/off系统。
2019年德克萨斯大学奥斯汀分校的Lacagnina等[29]使用Arc (activity-regulated cytoskeleton-associated protein)-CreERT2小鼠(Arc为即早基因, 在神经元激活时表达, 为神经元激活的标志物), 实验开始前向海马齿状回(dentate gyrus, DG)区显微注射携带激活和抑制性光敏感离子通道蛋白的AAV, 此时CreERT2融合蛋白虽已转录、翻译成蛋白质, 但未能入核, 故光敏感离子通道蛋白仍未表达。分别在进行恐惧记忆习得训练和恐惧记忆消退训练时注射4-OHT, 从而使在这2个不同实验过程中激活的神经元(分别称为恐惧获得神经元与恐惧消退神经元)特异性表达靶基因, 并在后续的实验中利用光遗传学人为激活、抑制靶神经元, 从而发现恐惧获得神经元在恐惧记忆的复发中起重要作用, 而恐惧消退神经元则抑制其复发。
2018年瑞士洛桑联邦理工学院Khalaf等[30]使用Tet-off系统采用c-fos-Cre小鼠进行实验。实验前向海马DG区显微注射激活性的DREADDs, 并持续给予Dox, 此时靶基因无法启动转录。在实验进行到恐惧记忆再提取时, 去除Dox, 在提取实验结束后继续给予Dox, 于是仅在恐惧记忆再提取过程中激活的神经元表达hM3Dq (为激活性DREADDs的一种), 从而进一步控制再提取中激活的神经元, 发现再提取激活的神经元与恐惧记忆的远期消退有密切关系。
CreERT2与Tet-on/off系统在恐惧记忆的应用提示, Cre前的启动子除了可以是组织特异性或神经元类型特异性的, 也可以是c-fos这类反应神经元激活状态的即早基因对应的启动子, 从而可以研究不同过程中激活的神经元的生理及病理作用。即使是同一核团、同一类型神经元在某一生理、病理过程中的作用也可能不同; 反之, 不同类型神经元在某一生理过程中的作用可能是一致或协同的。这种研究思路更具科学性与严谨性, 可能是今后神经科学研究中的趋势。
5 总结与展望
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Cre-loxP系统在组织、细胞特异性启动子的驱动下, 结合立体定位注射, 使该系统具有很高的组织特异性或空间特异性。在该系统的衍生系统中, CreERTtamoxifen系统在翻译后水平对Cre入核时间进行调控, Tet-on/off系统在转录水平上对Cre启动转录的时间进行调控。因此这2个衍生系统在具有空间特异性的基础上同时具备优良的时间特异性, 可以做到控制目的基因在何时、何处精确表达, 应用前景十分广阔。
同时也要注意到Cre-loxP系统存在一定的不足, 比如Cre、loxP纯合小鼠构建过程中是利用同源重组的原理, 不能排除导入的打靶DNA片段插入位置是否处于正确位置, 如果位置错误可能会导致其他基因的功能被破坏, 所以对筛选打靶DNA是否插入及插入位置均需要进行确认。其次, 表达出的Cre对宿主细胞可能也有一定影响, 如催化异常重组、抑制细胞增殖、促进细胞凋亡等[31]。因此研发性能更加优越的Cre与loxP序列突变体可能是优化Cre-loxP系统的发展方向之一。
作者贡献:张杨和贾林涛负责文献检索整理与论文撰写; 闫雨冬、赵亚男和张月明负责文献检索与资料提取; 杨素荣负责立题、整体设计和论文修改。
利益冲突:所有作者均声明不存在利益冲突。
基金
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  • 国家自然科学基金资助项目(81571296)
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2020年第55卷第9期
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doi: 10.16438/j.0513-4870.2020-0127
  • 接收时间：2020-02-14
  • 首发时间：2026-01-23
  • 出版时间：2020-09-12
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  • 收稿日期：2020-02-14
  • 修回日期：2020-03-28
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    1.复旦大学基础医学院, 上海 200032
    2.复旦大学基础医学院药理学系, 上海 200032

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