Abstract:

Genetically engineered mouse models remain central to studies of gene function, disease mechanisms, and preclinical drug development. Their generation has historically relied on two main routes: targeted modification through homologous recombination in embryonic stem cells (ESCs) and forward genetic approaches driven by random mutagenesis. In recent years, the landscape has shifted substantially with the introduction of programmable nuclease-based genome editing. Homologous recombination in ESCs provided the first reliable framework for precise genome manipulation, enabling the creation of constitutive and conditional knockouts as well as defined point mutations. However, this approach is often limited by lengthy procedures, high financial costs, and restricted applicability across different mouse strains. In contrast, random mutagenesis methods--such as N-ethyl-N-nitrosourea (ENU) treatment and transposon insertion-are better suited to large-scale forward screens due to their efficiency and scalability. The development of programmable nucleases, including zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cassystems, has markedly improved the efficiency and versatility of targeted genome editing. These technologies enable multiplexed genome modifications and the rapid generation of complex genotypes, thereby shifting mouse model production from sequential single-gene manipulation toward more scalable, multi-locus engineering. More recently, advanced derivatives such as base editing, prime editing, and RNA editing have further expanded the genome engineering tool box by reducing dependence on DNA double-strand breaks (DSBs). Such approaches enable single-nucleotide changes, small insertions or deletions, and transcript-level modulation with improved precision and flexibility. Despite these advances, unintended effects, including off-target mutations and genomic alterations-may still occur during founder (F0) generation and germline transmission. These risks can be reduced by using high-fidelity genome editing systems, comprehensive off-target analysis, multigenerational backcrossing, and in vivo functional rescue experiments. Taken together, the field of genetically engineered mouse research is progressing toward greater precision, standardization, and high-throughput capability, driven by continuous innovation in genome editing technologies. Careful consideration of editing efficiency, safety, and ethical constraints remains essential to ensure the robustness and reliability of generated models.

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