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Knockout mouse

A knockout mouse is a genetically engineered laboratory mouse (Mus musculus) in which researchers have inactivated, or "knocked out," one or more specific genes by replacing or disrupting them with an artificial DNA sequence, enabling the study of gene function and its effects on development, physiology, and disease.^[1] These mice serve as powerful tools in biomedical research, providing insights into the roles of individual genes within the mammalian genome.^[2] The creation of knockout mice typically involves embryonic stem (ES) cells derived from early mouse embryos, where gene targeting occurs through homologous recombination—a process that precisely inserts artificial DNA into the target gene locus—or gene trapping, which randomly inserts DNA to disrupt genes.^[1] Modified ES cells are then injected into blastocysts, implanted into surrogate mothers, and bred to generate chimeric mice, which are further crossed to produce homozygous knockout offspring with the gene fully inactivated in all cells.^[1] This technique, while efficient for known gene sequences, can result in embryonic lethality in about 15% of cases, requiring conditional or tissue-specific knockouts for viable models.^[1] The development of knockout mice traces back to foundational work in the 1980s, building on earlier discoveries like homologous recombination in bacteria (1958) and the isolation of mouse ES cells.^[2] Pioneered independently by Mario R. Capecchi, Martin J. Evans, and Oliver Smithies, the first gene-targeted knockout mice were reported in 1989, revolutionizing genetic research by allowing precise gene inactivation in mammals.^[2] Their contributions earned the Nobel Prize in Physiology or Medicine in 2007, recognizing the method's role—as of 2007—in elucidating over 10,000 genes (nearly half the mammalian genome) and creating more than 500 models of human disorders; large-scale projects like the International Mouse Phenotyping Consortium have since targeted nearly all protein-coding genes, expanding to approximately 13,000 strains as of 2024.^[2]^[3] Knockout mice have broad applications in modeling human diseases, including cancer, obesity, diabetes, heart disease, and neurological conditions like Parkinson's, by revealing how gene absence contributes to pathology.^[1] Notable examples include the p53 knockout mouse, which mimics Li-Fraumeni syndrome and aids cancer research, and strains like Methuselah for studying longevity or Frantic for anxiety disorders.^[1] Beyond disease modeling, they facilitate drug testing, therapy development, and understanding embryonic development, aging, and gene interactions, though phenotypic differences from humans sometimes limit direct translation.^[1]^[2]

Definition and Background

Definition

A knockout mouse is a genetically engineered laboratory mouse (Mus musculus) in which one or both alleles of a specific gene have been inactivated or disrupted, typically through targeted insertion of a selectable marker or disruption of the coding sequence, to study the gene's function.^[4] This modification is achieved via homologous recombination in embryonic stem cells, resulting in a null mutation that eliminates the gene's normal expression.^[5] The resulting mice often exhibit observable changes in phenotype, such as alterations in development, physiology, or behavior, which provide insights into the gene's role under normal conditions.^[4] Unlike transgenic mice, which involve the random insertion of additional genes to overexpress or introduce foreign DNA, knockout mice specifically inactivate endogenous genes without adding new genetic material.^[5] They also differ from knock-in mice, where a precise replacement or insertion of a modified gene sequence occurs at the target locus, rather than simple disruption.^[5] This targeted inactivation allows researchers to elucidate loss-of-function effects, distinguishing knockouts as a key tool for reverse genetics.^[6] Key terminology includes homozygous knockouts, where both alleles are inactivated, often leading to more pronounced phenotypes, and heterozygous knockouts, with only one allele disrupted, which may show subtler or no effects depending on the gene's dominance.^[4] Successful generation requires germline transmission, where the mutation is passed through the chimeric founder's germ cells to offspring, establishing a stable mutant line for breeding.^[5]

History

The development of knockout mouse technology began in the late 1970s and early 1980s with foundational advances in embryonic stem (ES) cell research. During this period, researchers established methods to isolate and culture pluripotent cells from mouse embryos, which proved essential for precise genetic manipulations. In 1981, Martin J. Evans and Matthew H. Kaufman reported the successful derivation of ES cell lines from the inner cell mass of mouse blastocysts, demonstrating their ability to contribute to all tissues in chimeric mice upon reimplantation. This breakthrough provided a cellular platform for introducing targeted genetic changes that could be transmitted through the germline.^[7] The pivotal advancement in gene targeting came through the application of homologous recombination, a natural DNA repair mechanism, to inactivate specific genes in mammals. In the early 1980s, Mario R. Capecchi and Oliver Smithies independently demonstrated that homologous recombination could be harnessed to modify genes in cultured mammalian cells with high specificity. Building on the ES cell technology pioneered by Evans, these methods were adapted to mouse ES cells, enabling the creation of the first knockout mice in 1989, where specific genes were deliberately disrupted to study their functions. This innovative approach revolutionized genetic research by allowing loss-of-function analysis in a whole-animal model. For their contributions to gene targeting in mice, Capecchi, Smithies, and Evans were awarded the 2007 Nobel Prize in Physiology or Medicine.^[8]^[2] By the mid-2000s, the field transitioned from targeted, labor-intensive gene knockouts to large-scale, systematic production efforts, driven by the completion of the mouse genome sequence and advances in automation. In 2006, the National Institutes of Health launched the Knockout Mouse Project (KOMP), a trans-NIH initiative aimed at generating targeted mutations in every protein-coding gene in the mouse genome using high-efficiency targeting vectors and ES cell screening. Complementing this, the International Knockout Mouse Consortium (IKMC) was established in 2007 through international collaboration, coordinating resources from multiple centers to produce and distribute knockout ES cell lines and mice for approximately 20,000 genes. These projects accelerated the generation of knockout models, shifting the focus toward comprehensive phenotyping and functional annotation of the mammalian genome. As of 2024, KOMP and the International Mouse Phenotyping Consortium (IMPC) have produced knockout strains for at least 9,700 genes, with ongoing efforts to target the remaining approximately 3,000 protein-coding genes.^[9]^[10]^[11]^[3]

Generation Methods

Traditional Gene Targeting

Traditional gene targeting, the foundational method for generating knockout mice, relies on homologous recombination to precisely disrupt a specific gene in the mouse genome. This technique, pioneered in the 1980s by researchers including Mario Capecchi, Martin Evans, and Oliver Smithies—who shared the 2007 Nobel Prize in Physiology or Medicine for their contributions—enables the creation of mice lacking functional expression of a target gene, facilitating the study of gene function. The process begins with the construction of a targeting vector, a engineered DNA molecule containing long stretches of homologous sequences (typically 5-10 kb homology arms) from the target gene locus, flanking a selectable marker such as the neomycin resistance gene (neo^r) to disrupt the gene upon integration. These homology arms ensure site-specific recombination, while the marker allows for selection of successfully modified cells; the vector is usually linearized before use to promote recombination.^[4] The targeting vector is introduced into mouse embryonic stem (ES) cells, derived from the inner cell mass of blastocysts, via electroporation, a method that delivers the DNA using electric pulses to transiently permeabilize the cell membrane. Following electroporation, cells are cultured under selective conditions: positive selection with G418 (geneticin) kills cells without the neo^r integration, while negative selection using agents like ganciclovir targets cells with random, non-homologous insertions by exploiting a herpes simplex virus thymidine kinase (HSV-tk) gene in the vector. Surviving clones are then screened for homologous recombinants using techniques such as Southern blotting to detect the expected restriction fragment length changes or PCR to amplify junction fragments specific to the targeted locus. This step identifies rare correctly targeted ES cell lines, as homologous recombination occurs in only about 1 in 10^3 to 10^6 electroporated cells, far less frequently than random integrations.^[4] To produce live knockout mice, positively identified ES cells are microinjected into wild-type mouse blastocysts, which are then implanted into the uterus of pseudopregnant foster mothers. The resulting chimeric offspring, with tissues derived from both ES and blastocyst cells, are bred to wild-type mice to transmit the modified allele through the germline. Heterozygous progeny are interbred to generate homozygous knockout mice, confirming the null mutation through genotyping and phenotypic analysis. Key challenges include off-target integrations that can disrupt unintended genomic regions and the inherently low efficiency of homologous recombination, necessitating screening of hundreds to thousands of ES clones per project.^[4] The entire process, from vector design to establishment of a homozygous knockout line, typically spans 1-2 years, reflecting the multi-step nature and breeding timelines involved.

Modern Genome Editing Techniques

Modern genome editing techniques have revolutionized the generation of knockout mice by enabling direct and efficient modifications in zygotes, bypassing the need for embryonic stem (ES) cell-based homologous recombination. The introduction of CRISPR-Cas9 in 2013 allowed for rapid gene disruption through the microinjection of Cas9 nuclease mRNA or protein along with a single guide RNA (sgRNA) designed to target specific genomic loci.00467-4) The sgRNA directs the Cas9 endonuclease to induce a double-strand break at the target site, which is predominantly repaired via non-homologous end joining (NHEJ), resulting in insertions or deletions (indels) that often cause frameshifts and premature stop codons, effectively knocking out the gene.00467-4) This one-step approach in fertilized oocytes produces founder mice carrying mutations in as little as 4 weeks, with high transmission rates to subsequent generations.00467-4) In comparison to traditional methods, CRISPR-Cas9 dramatically reduces the timeline and increases success rates for generating knockout mice. Conventional homologous recombination in ES cells typically requires 1-2 years to produce and validate targeted lines, involving lengthy steps like construct design, cell screening, and animal breeding.^[12] CRISPR, by contrast, achieves viable knockouts in weeks to months, with efficiencies reaching up to 100% for complete gene disruption in injected embryos in optimized protocols.^[13] This speedup is attributed to the direct zygote editing, which eliminates ES cell culturing and chimera formation, while multiplexing—targeting multiple genes simultaneously with separate sgRNAs—enables the creation of compound mutants in a single generation.00467-4) Prior to CRISPR-Cas9, zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) served as programmable nucleases for genome editing in mice, but their adoption was limited by design complexity. ZFNs, introduced in the early 2000s, fuse zinc finger proteins to the FokI nuclease domain for site-specific cuts, while TALENs, emerging around 2010, use bacterial TALE domains for DNA recognition paired with FokI.^[14] Both required laborious protein engineering for each target, contrasting with CRISPR's RNA-based simplicity, which relies on short sgRNAs that are easier and cheaper to synthesize.^[14] Although ZFNs and TALENs achieved knockouts via NHEJ with efficiencies of 10-50% in some cases, CRISPR's multiplexing capability and lower off-target effects in mammalian cells established its dominance for mouse model production.^[14] As of 2025, advances in derivative technologies like base editing and prime editing have further refined knockout strategies in mice by enabling precise disruptions without double-strand breaks. Base editors, such as cytosine base editors (CBEs) and adenine base editors (ABEs), fuse a catalytically impaired Cas9 (dCas9 or nCas9) with deaminases to convert C•G to T•A or A•T to G•C at targeted sites, allowing introduction of stop codons or disruptive mutations with efficiencies up to 50-70% in mouse zygotes.^[15] Prime editing, developed in 2019 and optimized for rodents, uses a prime editing guide RNA (pegRNA) and a reverse transcriptase-Cas9 fusion to install indels or substitutions via a single-strand template, achieving 20-60% editing rates in mouse embryos while minimizing unintended indels.^[15] These DSB-free methods reduce toxicity and mosaicism in founders, enhancing the precision of knockouts for complex genetic studies in mice.^[16]

Types and Strains

Types of Knockout Models

Knockout mouse models are categorized based on the extent, timing, and specificity of gene inactivation, allowing researchers to address limitations of global disruptions such as embryonic lethality or off-target effects.^[17] Constitutive knockout mice feature complete and permanent inactivation of the target gene in all cells from the earliest embryonic stages, resulting in a total loss-of-function phenotype throughout the organism's life.^[18] These models are generated through traditional homologous recombination or modern CRISPR-based methods to disrupt critical exons, providing insights into essential gene functions but often limited by compensatory mechanisms or viability issues.^[19] Conditional knockout models enable spatially or temporally restricted gene inactivation, overcoming the drawbacks of constitutive approaches by targeting specific tissues or developmental stages. The most widely used system is Cre-loxP recombination, where loxP sites flank essential gene exons (creating "floxed" alleles), and Cre recombinase expression—driven by tissue-specific promoters—excises the intervening DNA sequence.^[20] For example, floxed mice crossed with neuron-specific Cre lines allow brain-restricted knockouts without affecting other organs.^[21] This approach, pioneered in early applications like targeted oncogene activation, has become foundational for studying cell-type-specific roles. Inducible knockouts represent a refinement of conditional models, providing precise temporal control over gene inactivation, often in adult animals to mimic disease onset or avoid developmental confounds. A common method employs tamoxifen-activated Cre-ER recombinase fused to a modified estrogen receptor, which translocates to the nucleus only upon ligand binding, enabling on-demand recombination in floxed backgrounds.^[22] This system facilitates studies of gene function in mature tissues, such as cardiac-specific disruptions for adult-onset cardiomyopathy research.^[23] Partial or hypomorphic knockout models achieve reduced, rather than complete, gene function through subtle genetic alterations like point mutations or incomplete disruptions that lower expression or impair protein activity without full ablation.^[24] These alleles mimic human hypomorphic variants associated with partial loss-of-function diseases, offering viable models where null mutations are lethal, as seen in studies of metabolic disorders.^[25] Unlike knockouts, which permanently disrupt genomic DNA, knockdown approaches—typically using RNA interference (RNAi) via short hairpin RNAs—transiently reduce gene expression at the mRNA level without altering the genome, providing reversible but incomplete suppression suitable for short-term studies.^[26] In contrast, knock-in models involve precise insertion or replacement of genetic sequences, such as tagging a gene with a reporter or introducing human variants, rather than inactivation, to study gain-of-function or expression patterns.^[27]

Available Strains and Resources

Knockout mouse strains are primarily archived and distributed through major international repositories, including The Jackson Laboratory (JAX) in the United States, the European Mouse Mutant Archive (EMMA) as part of the INFRAFRONTIER infrastructure in Europe, and the Knockout Mouse Project (KOMP) repository managed by the Mutant Mouse Resource & Research Center (MMRRC). JAX holds thousands of mutant strains, facilitating broad access for research. EMMA cryopreserves and distributes over 8,900 mutant lines, emphasizing medically relevant models. The KOMP repository, an NIH initiative, offers more than 10,000 targeted knockout lines as of 2025, including embryonic stem (ES) cell lines, vectors, and live mice to support functional genomics studies.^[28]^[29] Strain nomenclature adheres to guidelines from the International Committee on Standardized Genetic Nomenclature for Mice, with the majority of knockout lines maintained on the C57BL/6 inbred background for genetic uniformity and reproducibility. Congenic strains, developed via repeated backcrossing (typically 10 generations), incorporate the knockout allele onto this background to reduce genetic drift and confounding variables from mixed origins; examples include C57BL/6J or C57BL/6N substrains, distinguished by their source or minor genetic differences.^[30] These repositories ensure strain maintenance through a combination of live breeding colonies for rapid distribution and cryopreservation techniques, such as freezing embryos or sperm, to safeguard against loss and enable long-term storage. Associated phenotyping data are centralized in resources like the International Mouse Phenotyping Consortium (IMPC) database, which provides standardized phenotypic information for over 8,700 genes as of early 2025, with ongoing efforts to cover the approximately 20,000 protein-coding genes in the mouse genome and aiming for 11,846 lines by 2027.^[31]^[32]^[33]^[34] Access to strains occurs via these resource centers, often requiring material transfer agreements to track usage and ensure biosafety. Distribution costs for non-profit and academic users vary by format and repository; for example, cryopreserved sperm from MMRRC costs $459 per line, while embryo revival to produce a litter ranges from $2,590, and comprehensive strain procurement—including shipping, health testing, and initial breeding setup—can total $5,000 to $20,000 depending on complexity. NIH-funded open-access initiatives, such as those under KOMP, subsidize or waive fees for ES cells and select strains to promote equitable research access worldwide.^[35]^[36]^[3]

Applications

Basic Research Uses

Knockout mice have been instrumental in studying loss-of-function phenotypes to infer the roles of specific genes in fundamental biological processes, such as embryogenesis, metabolism, and behavior. By disrupting a target gene, researchers observe the resulting abnormalities, which reveal the gene's normal contributions to development and physiology. For instance, the p53 knockout mouse, generated by homologous recombination to delete the Trp53 gene, demonstrated that p53 is essential for maintaining genomic stability, as homozygous mutants developed normally without overt developmental defects, but are prone to spontaneous tumor development in adulthood.^[37] This approach has similarly elucidated roles in metabolic pathways, where knockouts of genes like Insr (insulin receptor) display impaired glucose homeostasis and altered energy expenditure, highlighting insulin signaling's integration with lipid metabolism. In forward genetics, systematic screening of knockout lines through initiatives like the International Mouse Phenotyping Consortium (IMPC) assigns functions to previously uncharacterized genes by cataloging phenotypes across standardized assays. The IMPC has produced and phenotyped over 10,000 knockout lines as of 2024, identifying novel gene roles in processes like sensory perception and skeletal development through high-throughput behavioral, physiological, and anatomical evaluations.^[38]^[34] For example, phenotyping efforts have revealed embryonic lethality and disruptions in neural tube closure for certain genes, linking them to essential developmental pathways.^[39] This resource enables unbiased discovery, prioritizing genes based on phenotypic severity to map functional networks, with ongoing efforts aiming to phenotype approximately 60% of the mouse genome by 2027. Comparative studies involving crosses of knockout mice with other mutants dissect complex pathways, such as signaling cascades in immune responses, by isolating epistatic interactions. Double-mutant analyses, like those combining RelA (p65) and c-Rel knockins, have traced NF-κB activation dynamics in immune cells, showing how subunit-specific disruptions alter cytokine production and T-cell differentiation without complete pathway ablation.^[40] Such crossings reveal pathway hierarchies; for instance, Nfkb1/Nfkb2 double knockouts impair B-cell maturation and antigen receptor signaling, delineating non-canonical NF-κB's role in lymphoid organogenesis.^[41] Beyond core physiology, knockout mice provide insights into non-disease contexts like evolution, aging, and environmental interactions. Evolutionary studies use knockouts to probe conserved mechanisms, such as Hox gene disruptions that recapitulate ancestral patterning defects, illustrating how gene loss mimics speciation events in vertebrate limb development.^[42] In aging research, models like telomerase-deficient knockouts (Terc-/-) exhibit progressive telomere shortening, accelerating cellular senescence and revealing DNA damage responses as drivers of age-related tissue dysfunction.^[43] For environmental interactions, genotype-environment studies in knockout strains, such as serotonin receptor knockouts exposed to varying social housing, demonstrate how genetic backgrounds modulate behavioral plasticity, underscoring gene-by-environment effects on neuroplasticity.^[44] Strains from repositories like The Jackson Laboratory facilitate these investigations by providing standardized genetic backgrounds.^[28]

Disease Modeling and Therapeutics

Knockout mice have been pivotal in modeling human genetic disorders by recapitulating disease phenotypes through targeted gene inactivation. For instance, CFTR knockout mice exhibit intestinal obstruction, inflammation, and impaired mucociliary clearance, mirroring key aspects of cystic fibrosis pathology in humans.^[45] Similarly, ApoE knockout mice demonstrate accelerated amyloid plaque deposition and cognitive deficits when crossed with amyloid precursor protein transgenics, providing insights into Alzheimer's disease mechanisms and the role of apolipoprotein E in amyloid-beta aggregation.^[46] These models enable detailed study of disease progression and have informed therapeutic strategies for monogenic conditions. In cancer research, knockout mice targeting oncogenes or tumor suppressors have elucidated tumorigenesis and metastasis pathways. Inactivation of tumor suppressor genes like p53 or Rb in mice leads to spontaneous tumor formation, rapid progression, and metastatic spread, allowing researchers to dissect the multistep nature of carcinogenesis and evaluate interventions such as chemotherapy or immunotherapy.^[47] For example, p53 knockout models reveal how loss of this guardian gene promotes genomic instability and resistance to apoptosis, contributing to a deeper understanding of oncogene-driven cancers like those in the lung and breast.^[48] Knockout mice facilitate drug discovery through high-throughput phenotypic screening, where compounds are tested for their ability to rescue disease-like traits. In obesity models, leptin receptor knockout (db/db) mice display hyperphagia, insulin resistance, and metabolic syndrome, serving as platforms for screening anti-obesity agents that modulate energy balance or glucose homeostasis.^[49] These efforts have identified novel targets and validated therapies, with genome-wide knockout screens linking over 200 genes to body fat regulation.^[50] In therapeutic development, knockout models validate gene therapy targets by assessing correction of phenotypes, as seen in preclinical studies for neuromuscular and metabolic disorders.^[51] Knockout mice have contributed to the development of therapeutics across oncology, neurology, and rare diseases, enhancing translation to clinical trials.^[52] Their relevance to human precision medicine is evident in engineering patient-derived mutations, enabling personalized evaluations of genotype-phenotype relationships and trial designs.^[53] Conditional knockout approaches further refine these models for tissue-specific or temporally controlled disease simulation.^[54]

Limitations and Challenges

Biological and Technical Limitations

One significant biological limitation of knockout mice arises from embryonic lethality, where approximately 15% of gene knockouts result in developmentally lethal embryos that fail to progress to adulthood.^[1] This high rate of in utero death, often due to essential roles of the targeted gene in early development, restricts direct study of gene function in viable adult models and necessitates the use of conditional knockout strategies, such as Cre-loxP systems, to bypass lethality by enabling tissue- or time-specific gene inactivation.^[55] Another inherent biological challenge is genetic compensation, whereby the knockout of a target gene triggers upregulation of related genes, particularly paralogs, which can mask or alter expected phenotypes through functional redundancy.^[56] For instance, in cases of paralog redundancy, closely related genes sharing sequence and functional similarity may increase expression to compensate for the loss, leading to subtler or absent phenotypes than anticipated and complicating interpretation of gene function.^[57] Knockout mice also face limitations from species-specific physiological differences compared to humans, which hinder translational relevance in disease modeling. Mice exhibit a much shorter lifespan—typically 2-3 years versus over 70 years in humans—impacting the study of chronic conditions like neurodegeneration or cancer progression. Additionally, differences in immune system architecture, such as higher lymphocyte proportions and distinct T cell responses, result in divergent immune reactions to pathogens and therapies, reducing the predictive value for human outcomes.^[58] On the technical side, CRISPR/Cas9-mediated knockouts in mice are prone to off-target effects, where the Cas9 nuclease cleaves unintended genomic sites due to guide RNA mismatches, with reported mutation frequencies ranging from 0.01% to 1% depending on guide design and target sequence. These effects, though mitigated by high-fidelity Cas9 variants and optimized guide RNAs, can introduce confounding mutations that alter phenotypes or viability. Mosaicism further complicates founder generation, as CRISPR editing via pronuclear injection often yields embryos with heterogeneous genotypes across cells, observed in nearly all founders and leading to inconsistent germline transmission and unreliable initial phenotyping.^[59]^[60] Phenotypic variability in knockout mice is exacerbated by environmental and epigenetic factors, which can modify gene expression and outcomes beyond the genetic alteration alone. External influences like diet, stress, or housing conditions interact with epigenetic mechanisms, such as DNA methylation, to produce inconsistent phenotypes across litters or even within individuals, underscoring the need for standardized experimental controls to isolate genotypic effects.^[61] Advances in CRISPR guide design and delivery methods have begun to address some of these technical constraints by lowering off-target rates and mosaicism.

Ethical and Practical Considerations

Knockout mouse research raises significant ethical concerns centered on animal welfare, with the 3Rs principle—replacement, reduction, and refinement—serving as a foundational framework to minimize harm and optimize scientific outcomes.^[62] Replacement involves seeking non-animal alternatives where possible, reduction aims to decrease the number of animals used through improved experimental design, and refinement focuses on enhancing procedures to lessen pain and distress.^[63] In the United States, Institutional Animal Care and Use Committees (IACUCs) oversee compliance, requiring protocols that incorporate the 3Rs and ensuring humane treatment during breeding, phenotyping, and experimentation.^[64] Ethical debates surrounding knockout mice often highlight tensions between their value in disease modeling and broader implications for human gene editing technologies like CRISPR-Cas9. While knockout models provide critical insights into gene function and pathology, their creation parallels germline editing techniques, raising concerns about unintended off-target effects and the ethical slippery slope toward human applications.^[65] For instance, the ease of generating knockouts in mice has fueled discussions on germline modifications, as seen in post-CRISPR controversies where animal studies inform human trials but risk normalizing heritable changes without adequate safeguards.^[66] Practically, generating custom knockout mouse lines remains resource-intensive, with costs typically ranging from $8,000 to $25,000 or more as of 2025, depending on complexity and including CRISPR editing, validation, and colony establishment.^[67]^[68] Timelines have shortened to 3–4 months for founders using CRISPR, yet full strain development, including breeding and phenotyping, can extend to 6–12 months due to validation needs.^[69] Intellectual property restrictions further complicate strain sharing, as patents on engineered lines can limit access for academic researchers, prompting NIH policies to encourage deposition in public repositories while allowing institutions to retain invention rights.^[70]^[71] Regulatory oversight ensures ethical standards, with the U.S. National Institutes of Health (NIH) mandating IACUC review and, in 2025, prioritizing human-based alternatives through initiatives that de-emphasize animal-exclusive funding opportunities to promote broader methodological diversity. This initiative, announced in April 2025, shifts funding priorities toward human-based alternatives like organoids while allowing animal models in complementary roles, aiming to reduce overall animal use without eliminating necessary validation.^[72] In the European Union, Directive 2010/63/EU governs animal research, requiring severity assessments for genetically altered mice and emphasizing transparency in phenotyping data via recent regulations like (EU) 2019/1010, which enhance public reporting to support the 3Rs.^[73]^[74] As partial replacements, in vitro organoids derived from human induced pluripotent stem cells and computational models offer ways to study gene knockouts without whole-animal use, recapitulating tissue-level phenotypes and reducing reliance on mice for initial validation.^[75] These alternatives, while complementary rather than fully substitutive, align with ethical pushes to refine research by integrating multi-scale approaches that address biological limitations like species differences.^[76]

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The International Mouse Phenotyping Consortium (IMPC) is building a catalogue of mammalian gene function by producing and phenotyping a knockout mouse line ...
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Disease Model Discovery from 3,328 Gene Knockouts by The ...
Dec 26, 2017 · In conclusion, the IMPC has established an ever-expanding knowledgebase of mammalian gene function, a large resource of novel disease models and ...
[38]
Dissection of the NF-κB signalling cascade in transgenic ... - Nature
Feb 10, 2006 · Studies in transgenic and knockout mice have made a major contribution to our current understanding of the physiological functions of the NF-κB signalling ...
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Double knockin mice show NF-κB trajectories in immune signaling ...
The abundance trajectory revealed in our double knockin mice suggest that such tight regulation may have a broader role in immune cell development.
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Mouse models of human disease: An evolutionary perspective - NIH
The use of mice as model organisms to study human biology is predicated on the genetic and physiological similarities between the species.
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Mouse Models to Disentangle the Hallmarks of Human Aging
Sep 13, 2018 · In this review, we revisit these hallmarks with the information obtained exclusively through the generation of genetically modified mouse models.
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Genotype–environment interactions in mouse behavior: A way out of ...
Mar 22, 2005 · In behavior genetics, behavioral patterns of mouse genotypes, such as inbred strains, crosses, and knockouts, are characterized and compared ...
[43]
Pathophysiology of Gene-Targeted Mouse Models for Cystic Fibrosis
This paper reviews the pathophysiology in the tissues and organs (gastrointestinal, airway, hepatobiliary, pancreas, reproductive, and salivary tissue) ...
[44]
Apolipoprotein E is essential for amyloid deposition in the ... - PNAS
To determine whether apoE directly impacts amyloid deposition in vivo, we have crossed Apoe knockout mice with transgenic mice overexpressing a human mutant APP ...
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Mouse models for cancer research - PMC - NIH
Gene-Targeting Mouse Model of Cancer Using a knockout approach, the expression of tumor suppressor genes such as Rb and p53 as well as candidate tumor ...Mouse Models For Cancer... · Transgenic Mouse Model Of... · Gene-Targeting Mouse Model...
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Transgenic and Knockout Mice Models to Reveal the Functions of ...
In this review, we summarize some of the important transgenic and knockout mouse models for TSGs, including Rb, p53, Ink4a/Arf, Brca1/2, and their related ...
[47]
From Leptin to Lasers: The Past and Present of Mouse Models of ...
The use of obese mouse models provides unique insight into the hormones and mechanisms that regulate appetite and metabolism.
[48]
High-Throughput Screening of Mouse Gene Knockouts Identifies ...
Aug 28, 2021 · High-Throughput Screening of Mouse Gene Knockouts Identifies Established and Novel High ... Because obesity of a mouse gene knockout (KO) line ...Missing: drug discovery
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A review of animal models utilized in preclinical studies of approved ...
Apr 22, 2024 · This comprehensive review explores the use of animal models in preclinical gene therapy studies for approved products up to September 2023.
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https://pubmed.ncbi.nlm.nih.gov/34483672/
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Mouse systems genetics as a prelude to precision medicine - PMC
... clinical trials. We review here the recent advances applying systems ... Research: C57BL/6 and the International Knockout Mouse Consortium. Diabetes ...
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The role of mouse tumour models in the discovery and development ...
Jun 24, 2019 · However, the translation of drug efficacy in these models into outcomes in clinical trials ... knockout chimeric mice produced via blastocyst ...<|control11|><|separator|>
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Rescuing Lethal Phenotypes Induced by Disruption of Genes in Mice
Approximately 35 % of the mouse genes are indispensable for life, thus, global knock-out (KO) of those genes may result in embryonic or early postnatal ...
[54]
Genetic compensation: A phenomenon in search of mechanisms
Jul 13, 2017 · Upregulation of related genes following a gene knockout may be a direct consequence of the loss of protein function. For example, mice lacking ...
[55]
Functional Compensation of Mouse Duplicates by their Paralogs ...
Aug 10, 2022 · Our results imply that duplicates can often compensate for the loss of their paralogs, but only if they are expressed in the same tissues.Results · Gene Age Does Not Explain... · Sequence Similarity With The...
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The Applicability of Mouse Models to the Study of Human Disease
This chapter explores the most salient features of mouse models of human disease and provides a full assessment of the advantages and limitations of these ...
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Off-target Effects in CRISPR/Cas9-mediated Genome Engineering
Here, we review the basic mechanisms underlying off-target cutting in the CRISPR/Cas9 system, methods for detecting off-target mutations, and strategies for ...
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Pervasive Genotypic Mosaicism in Founder Mice Derived from ... - NIH
Jun 8, 2015 · The discovery of genotypic differences between tail lysate and germline transmission suggests prevalent mosaicism in founder mice derived from ...
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Epigenetics and phenotypic variability: some interesting insights ...
Jun 11, 2013 · Most economically relevant traits in animal production exhibit continuous phenotypic variations due to polygenic and environmental factors.
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Ethical Considerations in Animal Research: The Principle of 3R's
The significance of experimental research, the main functions of IACUC, and the principle of the three R's (replacement, reduction, and refinement) are ...
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Ethical considerations regarding animal experimentation - PMC - NIH
... principle of the 3 Rs. The 3Rs referred to are Reduction, Refinement and Replacement, and are applied to protocols surrounding the use of animals in research.
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Ethical Considerations in Animal Research: The Principle of 3R's
The IACUC helps to optimize the use of lab animals, ensuring that the experiments are carried out properly and in a humanitarian manner. One of the functions of ...
[63]
CRISPR ethics: moral considerations for applications of a powerful tool
An efficient method for generation of knockout human embryonic stem cells using CRISPR/Cas9 System. ... gene knockouts; analysis of an aggrecan knockout cell line ...
[64]
Ethical considerations of gene editing and genetic selection
May 29, 2020 · ... knockout mice were created. Targeted gene editing was further ... When undertaking to knock out a gene in an embryo, it is vital to ...
[65]
Cost of Custom Mouse Model Generation: KO vs. KI vs. Conditional
Apr 29, 2025 · The cost of generating a KO model primarily involves the design and synthesis of the targeting vector, embryonic stem (ES) cell culture and ...
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Transgenic & Gene Targeting Mouse Core Facility - HSC Cores
The total cost to generate a novel mouse model from start to the sequenced heterozygous N1 generation is $8,000-$16,000 and depends on the mouse strain and edit ...
[67]
What Is The Mouse Knockout Timeline? - Ingenious Blog
Feb 18, 2021 · Today a founder mouse for a new knockout line can be generated in as little as 3 months. The pups of that founder can be genotyped 3-4 ...
[68]
Issues Posed by the New Mouse Genetics, and Possible Solutions
Three key subjects of concern are intellectual-property rights, safe and efficient distribution of the mice to researchers, and the handling of the ...
[69]
Model Organism Sharing Policy - NIH Grants & Funding
Aug 5, 2025 · Intellectual Property The investigator and their institution may choose to retain title to subject inventions such as a mutant mouse developed ...
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How Does the NIH Initiative to Prioritize Human-Based Research ...
Jul 18, 2025 · In July 2025, NIH announced it will no longer develop new funding opportunities focused exclusively on animal models of human disease.
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Protection of laboratory animals | EUR-Lex - European Union
Directive 2010/63/EU sets out measures designed to protect animals used for scientific purposes, including animals used in basic and applied research, ...
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Guidelines on severity assessment and classification of genetically ...
Jul 11, 2017 · The document serves as a guide to determine the degree of severity for an observed phenotype. The aim is to support scientists, animal care takers, animal ...
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Replacing the replacements: Animal model alternatives - Science
Oct 12, 2018 · New technologies—3D cell culturing, human induced pluripotent stem cells, and gene editing—are leading to new solutions for replacing, refining, ...
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Organoids are promising but not alternatives, yet – new feature
Jun 6, 2025 · Therefore, organoids should be seen as complementary to animal models rather than alternatives. Used together, these tools can help answer ...