基因工程小鼠构建技术的演进、应用与展望

doi:10.12300/j.issn.1674-5817.2026.028

实验动物与比较医学 ›› 2026, Vol. 46 ›› Issue (5): 759-770.DOI: 10.12300/j.issn.1674-5817.2026.028

基因工程小鼠构建技术的演进、应用与展望

吴剑锋¹^,²(), 邵志强¹, 贺颖¹()(), 吴素琴¹()

^1. 厦门大学实验动物中心, 厦门 361102
^2. 传染病疫苗研发全国重点实验室, 翔安创新实验室, 厦门 361102

收稿日期:2026-01-28 修回日期:2026-04-24 出版日期:2026-10-25 发布日期:2026-10-16
通讯作者: 贺颖（1982—），女，博士，高级工程师，研究方向：实验动物学。E-mail： hey@xmu.edu.cn。ORCID： 0009-0006-7431-5381
吴素琴（1987—），女，博士，高级实验师，研究方向：实验动物学。E-mail： caca_china@xmu.edu.cn。ORCID： 0009-0008-5852-5914
作者简介:
吴剑锋（1988—），男，博士，讲师，研究方向：人类疾病动物模型。E-mail： wzqwjf0728@xmu.edu.cn。ORCID： 0000-0002-8992-2030
基金资助:
国家自然科学基金面上项目“p38信号通路在CD8+T细胞抗病毒感染中的作用机制研究”(32470944); 厦门市自然科学基金面上项目“p38信号通路调控浆细胞相关疾病进程的机制研究”(3502Z202373021); “坏死样凋亡在全身性炎症反应综合征中的作用研究”(3502Z202373004); 翔安创新实验室/传染病疫苗研发全国重点实验室科技项目“靶向p38信号通路调节疫苗免疫效果”(2023XAKJ0101033)

Evolution, Applications, and Perspectives of Technologies for Genetically Engineered Mouse Generation

WU Jianfeng¹^,²(), SHAO Zhiqiang¹, HE Ying¹()(), WU Suqin¹()

^1. Xiamen University Laboratory Animal Center, Xiamen 361102, China
^2. State Key Laboratory of Vaccines for Infectious Diseases, Xiang'an Biomedicine Laboratory, Xiamen 361102, China

Received:2026-01-28 Revised:2026-04-24 Published:2026-10-25 Online:2026-10-16
Correspondence to:
HE Ying (ORCID: 0009-0006-7431-5381), E-mail: hey@xmu.edu.cn;

摘要/Abstract

摘要：

基因工程小鼠是解析基因功能、阐明疾病机制及推动药物研发的重要工具。随着生命医学研究对复杂遗传修饰、快速模型构建及高通量功能解析需求的不断增加，基因工程小鼠构建技术也在持续发展。本文系统梳理了基因工程小鼠构建技术的演进与应用，重点介绍以早期转基因和胚胎干细胞（embryonic stem cell，ESC）同源重组基因打靶为代表的经典构建技术，以及N-乙基-N-亚硝基脲（N-ethyl-N-nitrosourea，ENU）诱变、转座子插入等随机诱变技术在正向遗传学筛选中的应用；进一步阐述以锌指核酸酶、转录激活因子样效应物核酸酶及CRISPR/Cas系统为代表的可编程核酸酶在基因工程小鼠构建中的原理、特点及应用。随着基因组编辑技术的发展，Cas9衍生系统、碱基编辑、引导编辑及RNA编辑等技术进一步提高了编辑精度，拓展了遗传修饰与精准调控方式，并丰富了基因工程小鼠构建的技术选择。本文还比较了不同构建技术的优势、局限性及适用场景，并针对脱靶效应、嵌合性、遗传背景及遗传稳定性等影响模型可靠性的实践问题，归纳了相应的质量控制策略。最后，本文展望了基因工程小鼠构建技术的发展趋势，以期为相关模型的构建与应用提供参考。

关键词: 基因工程小鼠, 基因打靶技术, 随机诱变模型, CRISPR/Cas系统

Abstract:

Genetically engineered mice are important tools for elucidating gene function and disease mechanisms and for advancing drug development. With the growing demand in biomedical research for complex genetic modifications, rapid model generation, and high-throughput functional analysis, technologies for generating genetically engineered mice continue to evolve. This review systematically summarizes the evolution and applications of these technologies, with a focus on conventional approaches, including early transgenic technologies and embryonic stem cell (ESC)-based homologous recombination for gene targeting, as well as the use of random mutagenesis approaches, such as N-ethyl-N-nitrosourea (ENU) mutagenesis and transposon insertion, in forward genetic screening. It further discusses the principles and characteristics of programmable nucleases, including zinc finger nucleases, transcription activator-like effector nucleases, and CRISPR/Cas systems, as well as their applications in generating genetically engineered mice. With advances in genome editing, Cas9-derived systems, base editing, prime editing, and RNA editing further improve editing precision, expand the repertoire of genetic modifications and precise regulatory approaches, and provide additional options for generating genetically engineered mice. This review also compares the advantages, limitations, and suitability of different technologies for specific applications and summarizes quality-control strategies addressing practical issues that may affect model reliability, including off-target effects, mosaicism, genetic background, and genetic stability. Finally, future directions in technologies for generating genetically engineered mice are discussed to inform the generation and application of related models.

Key words: Genetically engineered mice, Gene targeting technology, Random mutagenesis models, CRISPR/Cas system

中图分类号:

R-332
Q95-33

吴剑锋,邵志强,贺颖,等. 基因工程小鼠构建技术的演进、应用与展望[J]. 实验动物与比较医学, 2026, 46(5): 759-770. DOI: 10.12300/j.issn.1674-5817.2026.028.

WU Jianfeng,SHAO Zhiqiang,HE Ying,et al. Evolution, Applications, and Perspectives of Technologies for Genetically Engineered Mouse Generation[J]. Laboratory Animal and Comparative Medicine, 2026, 46(5): 759-770. DOI: 10.12300/j.issn.1674-5817.2026.028.

图/表 2

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技术类型 Technology	编辑原理 Editing principle	是否依赖DSB Depen-dence on DSB	可实现的突变类型 Types of mutations achievable	编辑效率 Editing efficiency	构建周期 Model genera-tion time	构建成本 Cost	品系适应性 Strain compat-ibility	多基因编辑能力 Multiplex editing capability	脱靶风险 Off-target risk	主要优势 Key advantage	主要局限 Key limitation	典型应用 Typical application
胚胎干细胞同源重组 ESC-based homologous recombination	同源重组介导靶位点定向替换	否	KO、KI、点突变等均可实现	低	长（6～12个月）	高	受限（前期主要为129等品系）	低	低	精确性高，遗传结果稳定	周期长、成本高	精确遗传修饰、经典KO/KI模型
ENU/转座子诱变 ENU/Transposon mutagenesis	ENU诱导碱基烷基化引起点突变；转座子在基因组中随机或偏好性插入导致基因功能破坏	否	点突变（ENU）、插入突变（转座子）	高（突变率高）	长	中	高	高（但随机性强）	不适用于传统脱靶概念，且存在随机突变背景	无须预设靶点，可进行正向遗传筛选	位点鉴定困难、表型归因复杂	正向遗传筛选、新基因发现
锌指核酸酶 Zinc finger nucleases	蛋白识别DNA序列并由FokⅠ诱导产生DSB	是	KO、KI、点突变等均可实现	中	中	高	高	低	中	开创性定点编辑工具	设计复杂、成本高、专利限制	特定靶点编辑或已有体系延续
转录激活因子样效应物核酸酶 Transcription activator-like effector nuclease	转录激活因子样效应物识别DNA序列并由FokⅠ诱导产生DSB	是	KO、KI、点突变等均可实现	中～高	中	中～高	高	低	低	特异性较高、识别规则明确	构建复杂、递送受限	高特异性需求或复杂靶点编辑
CRISPR/Cas系统 CRISPR/Cas system	sgRNA引导Cas9在靶位点产生DSB	是	KO、KI、点突变等均可实现	高	短（2～3个月）	低	高	高（多条sgRNA实现多靶向）	中	灵活、高效、适用于多基因编辑	存在脱靶及嵌合性风险	常规基因工程小鼠构建首选
碱基编辑 Base editing	脱氨酶介导碱基转换	否	主要为点突变	高	短	中	高	中	低～中	无DSB、高精度	编辑窗口受限	单碱基突变疾病模型
引导编辑 Prime editing	逆转录酶介导的靶位点序列改写	否	片段序列精准插入/缺失/替换	中	短～中	中	高	中	低	突变类型多样、灵活性高	编辑效率相对较低	复杂突变或非编码区精细编辑模型

技术类型 Technology	编辑原理 Editing principle	是否依赖DSB Depen-dence on DSB	可实现的突变类型 Types of mutations achievable	编辑效率 Editing efficiency	构建周期 Model genera-tion time	构建成本 Cost	品系适应性 Strain compat-ibility	多基因编辑能力 Multiplex editing capability	脱靶风险 Off-target risk	主要优势 Key advantage	主要局限 Key limitation	典型应用 Typical application
胚胎干细胞同源重组 ESC-based homologous recombination	同源重组介导靶位点定向替换	否	KO、KI、点突变等均可实现	低	长（6～12个月）	高	受限（前期主要为129等品系）	低	低	精确性高，遗传结果稳定	周期长、成本高	精确遗传修饰、经典KO/KI模型
ENU/转座子诱变 ENU/Transposon mutagenesis	ENU诱导碱基烷基化引起点突变；转座子在基因组中随机或偏好性插入导致基因功能破坏	否	点突变（ENU）、插入突变（转座子）	高（突变率高）	长	中	高	高（但随机性强）	不适用于传统脱靶概念，且存在随机突变背景	无须预设靶点，可进行正向遗传筛选	位点鉴定困难、表型归因复杂	正向遗传筛选、新基因发现
锌指核酸酶 Zinc finger nucleases	蛋白识别DNA序列并由FokⅠ诱导产生DSB	是	KO、KI、点突变等均可实现	中	中	高	高	低	中	开创性定点编辑工具	设计复杂、成本高、专利限制	特定靶点编辑或已有体系延续
转录激活因子样效应物核酸酶 Transcription activator-like effector nuclease	转录激活因子样效应物识别DNA序列并由FokⅠ诱导产生DSB	是	KO、KI、点突变等均可实现	中～高	中	中～高	高	低	低	特异性较高、识别规则明确	构建复杂、递送受限	高特异性需求或复杂靶点编辑
CRISPR/Cas系统 CRISPR/Cas system	sgRNA引导Cas9在靶位点产生DSB	是	KO、KI、点突变等均可实现	高	短（2～3个月）	低	高	高（多条sgRNA实现多靶向）	中	灵活、高效、适用于多基因编辑	存在脱靶及嵌合性风险	常规基因工程小鼠构建首选
碱基编辑 Base editing	脱氨酶介导碱基转换	否	主要为点突变	高	短	中	高	中	低～中	无DSB、高精度	编辑窗口受限	单碱基突变疾病模型
引导编辑 Prime editing	逆转录酶介导的靶位点序列改写	否	片段序列精准插入/缺失/替换	中	短～中	中	高	中	低	突变类型多样、灵活性高	编辑效率相对较低	复杂突变或非编码区精细编辑模型

基因工程小鼠构建技术的演进、应用与展望

Evolution, Applications, and Perspectives of Technologies for Genetically Engineered Mouse Generation

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