Avoiding Common Pitfalls in Mouse Model Selection

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Avoiding Common Pitfalls in Mouse Model Selection

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Avoiding Common Pitfalls in Mouse Model Selection

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Selecting the right mouse model is a critical decision that can shape the success of your research. With the wide variety of available models, each suited to different experimental needs, researchers often face challenges in matching their study objectives with the appropriate mouse strain. Common pitfalls, such as ignoring genetic backgrounds, overlooking sex differences, or overusing immunocompromised mice, can lead to skewed results and misinterpretation of data. This blog will guide you through these pitfalls and provide practical tips for making informed decisions in mouse model selection, ensuring your research remains robust and translatable.

1. Understanding Experimental Goals

Pitfall: Selecting a mouse model without a clear understanding of the experimental objectives can lead to suboptimal study designs, irrelevant results, and wasted resources. Each mouse model is tailored to specific research questions, such as studying disease mechanisms, testing drug efficacy, or exploring genetic functions. Without aligning the model to the goal, critical factors like immune responses, genetic backgrounds, and physiological relevance may be overlooked, compromising the study’s validity.

Tips:

  • Define the Study’s Core Objective: Determine whether your primary aim is to explore disease pathogenesis, evaluate drug responses, or manipulate genes. For instance, if the goal is to study genetic mutations and their effects, inbred strains like C57BL/6 may be appropriate due to their genetic uniformity. For immunotherapy studies, models like humanised mice or syngeneic models are more suitable due to their immune compatibility.
  • Match Models to Objectives:
    • Disease Mechanism Studies: Use genetically engineered models (knockout, transgenic) that closely mimic human disease conditions, allowing insights into underlying mechanisms.
    • Drug Response Evaluation: Outbred or genetically diverse models, such as Diversity Outbred mice, help capture variability in treatment effects, reflecting more realistic drug responses.
    • Genetic Manipulation Studies: Models like the Cre-Lox system enable precise control over gene expression, helping dissect specific genetic contributions to disease.
  • Consider Translational Relevance: Select models that closely reflect human conditions relevant to your research. For studying MASH pathogenesis, ob/ob or db/db mice with leptin pathway mutations mimic obesity, insulin resistance, and liver inflammation. Methionine and choline-deficient (MCD) diet-fed mice are ideal for investigating liver inflammation and fibrosis without obesity, focusing on immune responses. For evaluating drug responses in MASH, the STAM model (streptozotocin and high-fat diet) effectively replicates human disease, including steatosis, fibrosis, and hepatocellular carcinoma.

*Check out Atlantis Bioscience’s solutions for MASH models.

Non-Alcoholic Steatohepatitis (NASH) Mouse Models – Atlantis Bioscience Pte Ltd

Non-alcoholic Steatohepatitis (NASH) Models l GemPharmatech

There is a change in the name for these mouse models from NASH to MASH. Can you help to update our website with the new information from GPT? I have attached the websites. 

By aligning mouse model selection with experimental objectives, researchers can enhance study design, ensure relevant data collection, and ultimately produce findings that are more applicable to human health.

2. Choosing Between Inbred vs. Outbred Strains

Pitfall: Misunderstanding the genetic variability between inbred and outbred strains can lead to inappropriate model selection, skewing results and limiting the generalisability of findings. Inbred strains, bred through brother-sister mating for over 20 generations, are genetically identical, whereas outbred strains maintain genetic diversity, more closely resembling the variability seen in human populations. Failing to appreciate these differences can affect reproducibility and the interpretation of experimental outcomes, especially in areas such as pharmacological studies or disease modelling.

Tips:

  • Inbred Strains: Inbred strains are highly consistent due to their genetic uniformity, making them ideal for experiments requiring high reproducibility. For example, if you’re studying the effects of a specific gene knockout or investigating fundamental biological processes where minimising variability is critical, inbred strains are the preferred choice. However, the homogeneity of inbred mice means that results may not fully capture how genetic variability influences biological responses in a more diverse population. For instance, a drug’s effect on an inbred mouse strain may differ significantly from its effect on a genetically diverse human population.
  • Outbred Strains: Outbred strains are genetically heterogeneous, providing a better model for studying drug responses, toxicology, and population-based traits where genetic diversity matters. These models are particularly useful when studying complex diseases influenced by multiple genetic and environmental factors, such as diabetes or cardiovascular disease. Outbred mice may also yield insights into how treatments might perform across a broader population, helping identify variability in responses that could be critical in clinical applications.

Table 1: Different Inbred and Outbred Mice

StrainDescription
Inbred Mice
C57BL/6A widely used inbred strain, particularly for transgenic and knockout models, obesity, and immunology research.  
BALB/cCommonly used for hybridoma development, monoclonal antibody production, infectious disease, and immunology studies.  
129/SvPreferred for genetic manipulation studies, especially in creating knockout mice.  
C3HUtilised in cancer, infectious disease, and cardiovascular research.  
Outbred Mice
CD-1Frequently used in genetics, toxicology, pharmacology, and aging research.  
Swiss WebsterPopular for general research, including safety and efficacy testing.  
ICRA versatile outbred model applicable in toxicity, pharmacology, drug efficacy, and immunology studies.  

By thoroughly understanding the genetic makeup of the strains used, researchers can avoid drawing inaccurate conclusions and ensure that the mouse model aligns with the study’s goals.

3. Ignoring Background Genetic Influences

Pitfall: Overlooking the genetic background of a mouse strain can significantly impact experimental results, particularly in genetically engineered models. A classic example is the difference in phenotype of the Lepob and Leprdb mutations on C57BL/6 vs. C57BLKS/J mice, where the mutations result in more severe obesity and diabetes phenotypes on the C57BLKS/J background. This highlights how background genetics can dramatically alter disease manifestation.

Tips:

  • Genetic Background Matters: Different strains have distinct genetic modifiers that can influence the severity, penetrance, and expressivity of phenotypes. For example, the widely used C57BL/6 strain may exhibit different immune responses compared to the BALB/c strain, affecting outcomes in immunological studies.

Table 2: Comparison between C57BL/6 and BALB/c Mice

CharacteristicC57BL/6 MiceBALB/c Mice
Innate Immune ResponsePredominantly Th1 immune responsePredominantly Th2 immune response
TregLessMore
Humoral ResponseWeaker humoral responseStronger humoral response
MHC Class I Gene LocusH2bH2d
Immune System CharacteristicsHigher IL-12 production; characterized by higher cytotoxic activity of splenic NK cellsIncreased T zone volume density in the spleen; production of IL-2, IL-3, IL-4, IL-10, and TNF-α
  • Choosing the Right Background: When developing genetically engineered models, such as knockouts or transgenics, carefully select the mouse strain that best matches your research goals.
  • Use of Congenic Strains: Congenic strains are created by transferring a specific gene or mutation onto a well-defined genetic background through repeated backcrossing. This approach helps isolate the influence of a specific gene against a consistent genetic backdrop, minimising background noise.

By paying attention to genetic background, researchers can better interpret the phenotypic effects of their models, ensuring that results are attributable to the targeted genetic modifications rather than unintended background influences.

4. Overlooking Gender Differences

Pitfall: Not considering sex differences in disease progression, physiology, or drug response can lead to skewed results and a lack of translatability to broader populations. Males and females can exhibit significant differences in immune function, metabolism, pain perception, and response to treatments. For instance, Jeffrey Mogil, a pain study specialist at McGill University, noticed that male mice became stressed around pregnant and lactating females due to a social signaling behaviour while the non-pregnant females in the neighbourhood are not stressed: the females’ urine contained compounds, similar to the smell of bananas, that warned males to stay away from their pups, causing stress in the males.

Tips:

  • Include Both Sexes in Study Design: Incorporate male and female mice in your experiments to capture a more comprehensive picture of the biological processes under study. This approach helps identify sex-specific responses and ensures that findings are not biased toward one sex, ultimately improving the relevance of your research to human health.
  • Analyse Data by Sex: Beyond just including both sexes, data should be analysed separately to detect sex-specific effects that could otherwise be masked when combined. For example, metabolic responses to a high-fat diet can vary greatly between male and female mice, impacting conclusions drawn about obesity or diabetes models.
  • Consider Hormonal Influences: Hormonal cycles, particularly in females, can influence experimental outcomes, such as immune responses or drug metabolism. Accounting for these variations by carefully timing experiments or adjusting analysis can provide deeper insights into sex-specific biology.

Addressing these differences enhances the reliability of preclinical models and supports the development of more targeted and effective therapeutic strategies for both men and women.

5. Overreliance on Immunocompromised Mice

Pitfall: Immunocompromised mice, such as nude or SCID mice, are widely used in cancer studies because they lack functional immune systems, allowing for easy tumour grafting. However, these models fail to capture the critical role of the immune system in cancer progression, metastasis, and treatment response. This overreliance can lead to misleading results, particularly when studying immunotherapies, where the interaction between tumours and the immune system is essential.

Tips:

  • Match Models to Immune Study Needs: When studying therapies that involve immune responses, such as checkpoint inhibitors or CAR-T cell therapies, consider using humanised mice—models that have been engrafted with a functional human immune system. These models provide a closer approximation of how human tumours interact with the immune system, enabling more accurate evaluation of immune-oncology treatments.
  • Assessing Tumour-Immune Interactions: In studies focused on immune mechanisms, syngeneic mouse models (where tumours are implanted into immunocompetent mice of the same genetic background) are valuable for exploring tumour-immune dynamics. They preserve a functional immune system, allowing researchers to study immune responses in a more natural context.
  • Exploring Limitations of Immunocompromised Models: While immunocompromised mice are invaluable for initial tumour growth studies and xenograft models, they do not reflect the complexities of the immune system’s role in cancer control. Researchers must balance the simplicity and convenience of these models with their limitations, selecting more sophisticated models when studying interactions between cancer and the immune system.

Table 3: Mouse models with intact immune system

CharacteristicSyngeneic Mouse ModelsImmune-Reconstituted Mouse Models
DefinitionModels using genetically identical mice of the same immunocompetent strain.Models involving the transfer of human immune cells into immunocompromised mice.
Types of Immune Cells UsedN/A (uses the mouse own immune response)– PBMC (Peripheral Blood Mononuclear Cells): Contains various immune cells, including T cells and B cells. – CD34+ Cells: Hematopoietic stem cells that can differentiate into various blood cell types. – BLT (Bone Marrow-Liver-Thymus): Contains human foetal liver and thymus, enabling the development of a more complete human immune system.
ApplicationsStudying tumour growth, metastasis, and the efficacy of immunotherapies.Studying human-specific immune responses, vaccine efficacy, and disease mechanisms.
Advantages– Reduces variability due to genetic homogeneity. – Useful for studying immune interactions within the same species.– Provides insights into human immune responses.
– Allows for the evaluation of therapies targeting human-specific pathways.
Limitations– Cannot study human immune responses directly.
– Limited to the interactions of mouse immune cells.		– Variability in reconstitution success.
– Immune responses may not fully mimic human responses due to the host environment.

By carefully selecting appropriate models that account for immune interactions, researchers can improve the translatability of their findings and develop more effective cancer therapies.

6. Assuming All Transgenic Models Are the Same

Pitfall: One common misconception in research is the belief that any transgenic mouse model can be applied interchangeably to address a specific research question. This assumption overlooks the inherent complexities and variations that exist among transgenic models, which can significantly influence experimental outcomes and interpretations. Each transgenic mouse model is designed with specific genes inserted into its genome, and these insertions can vary widely in terms of their location, the regulatory elements involved, and the overall genetic background of the host mouse strain.

Key Considerations

  1. Insertion Site Impact:
    • Gene Expression Levels: The position where a transgene is integrated into the mouse genome can affect its expression levels. For example, if a transgene is inserted near a transcriptionally active region, it may lead to higher expression levels compared to one inserted in a repressive chromatin environment.
    • Influence on Nearby Genes: The insertion of a transgene can inadvertently affect the expression of neighbouring genes, leading to unintended phenotypic changes or compensatory mechanisms that can confound the results of a study.
  2. Transgene Design:
    • Promoter Selection: The choice of promoter driving the transgene expression plays a critical role in determining the timing and level of gene expression. For instance, tissue-specific promoters will limit expression to particular organs, which may be essential for studying localised biological processes.
    • Vector Backbone: Different vectors used for transgene construction may have distinct characteristics that influence stability, expression duration, and the transgene’s integration efficiency.
  3. Mouse Strain Background:
    • Genetic Variability: The genetic background of the host strain can also significantly impact how the inserted transgene behaves. Different strains may respond differently due to variations in immune response, metabolic processes, or background gene interactions.
    • Phenotypic Effects: Even subtle differences in genetic background can lead to varying phenotypic expressions, making it critical to consider the specific strain used when interpreting results.

Tips:

To mitigate the risks associated with this pitfall, researchers should conduct a thorough review of the specific transgenic models under consideration:

  • Examine the Transgene Details: Investigate how the transgene was inserted into the mouse genome, including the insertion site and any accompanying regulatory elements. Understanding these factors will provide insights into potential variations in expression and function.
  • Literature Review: Consult relevant literature to identify previous studies using similar transgenic models, which can provide insights into how the insertion may influence outcomes and guide the design of new experiments.
  • Consult Experts: Collaborate with geneticists or molecular biologists who have experience with transgenic models to gain a deeper understanding of the implications of transgene design and background.

By recognising that not all transgenic models are created equal and by considering the specific details of each model, researchers can make more informed decisions in their experimental designs and interpretations, ultimately leading to more reliable and reproducible results.

7. Misinterpreting Phenotypes

Pitfall: One significant misconception in research involving mouse models is the misinterpretation of phenotypic changes. Subtle alterations in behaviuor, physical appearance, or physiological responses may be dismissed as insignificant or attributed to minor variability without thorough investigation. However, these seemingly minor changes can be indicative of deeper underlying biological issues, such as genetic mutations, environmental stresses, or improper experimental conditions. For instance, changes in activity levels, grooming behaviuors, or weight fluctuations can reflect serious health problems or altered biological pathways that require attention.

Key Considerations

  1. Behavioural Changes:
    • Assessment Methods: Many behavioural assays are used to evaluate mouse models, but the sensitivity and specificity of these methods vary. Subtle behavioural changes may not be easily captured by standard tests, leading researchers to overlook significant issues.
    • Contextual Factors: Environmental factors, such as stress from handling or changes in housing conditions, can also influence behaviour and may be mistakenly attributed to the experimental intervention rather than underlying health concerns.

Table 4: Factors that affect mouse behaviour

FactorDescription
Genetic BackgroundBehavioural traits vary significantly between strains, affecting learning, aggression, and anxiety levels.
SexMale and female mice often display different social, territorial, and stress responses.
AgeBehavioural patterns such as activity levels, cognitive function, and social interactions change with age.
EnvironmentHousing (e.g., cage size, bedding, enrichment) influences exploration, stress, and overall well-being.
Social StructureGroup housing versus isolation affects social behaviour, stress levels, and hierarchy dynamics.
HandlingFrequent or rough handling can cause anxiety, stress responses, or altered interactions.
Light CycleMice are nocturnal, so changes in light/dark cycles affect sleep, feeding, and activity patterns.
DietNutritional deficiencies or high-fat diets can influence behaviour, including aggression or anxiety.
TemperatureExtreme heat or cold can cause discomfort, altered activity, or stress-related behaviours.
Health StatusIllness, pain, or discomfort (e.g., from infection or injury) significantly impacts normal behaviour.
  1. Physical Appearance:
    • Phenotypic Variability: Normal phenotypic variation within and between strains can complicate the interpretation of results. For example, variations in coat colour, body size, or organ morphology might occur naturally and not necessarily indicate a pathological condition.
    • Health Indicators: Specific phenotypic traits, such as fur quality or posture, can be sensitive indicators of the overall health and well-being of the mice. Researchers should be vigilant in observing these traits and understand their implications.

Tips:

To avoid misinterpretation of phenotypes, researchers should implement a rigorous and systematic approach:

  • Conduct Detailed Phenotypic Analysis: Utilise comprehensive assessment protocols that include a variety of behavioural and physiological evaluations. This can involve using standardised tests and metrics to quantify behaviours and physical traits accurately.
  • Validate Unexpected Findings: When unexpected results arise, it is crucial to perform additional experiments or tests to confirm findings. This may include using control groups or alternative models to rule out confounding factors and ensure that observed changes are indeed significant and relevant.

By adopting these practices, researchers can improve their ability to detect and interpret subtle phenotypic changes, ultimately leading to more accurate conclusions about the underlying biology.

8. Failing to Consider Ethical and Practical Constraints

Pitfall: Another critical pitfall in mouse model research is the failure to consider ethical and practical constraints. Researchers may become so focused on their scientific objectives that they overlook the ethical implications of their work, including animal welfare concerns and the responsible use of resources. Additionally, logistical challenges such as housing, breeding, and availability of specific mouse strains can impact the feasibility of a study. Ignoring these factors can lead to significant challenges in the implementation and execution of research protocols.

Key Considerations

  1. Ethical Concerns:
    • Animal Welfare: Researchers must adhere to ethical guidelines governing the use of animals in research. This includes ensuring humane treatment, minimising suffering, and using the least number of animals necessary to achieve valid results. Ignoring these considerations can lead to ethical violations and damage the credibility of the research.
    • Regulatory Compliance: Compliance with institutional and governmental regulations is crucial. Researchers should be aware of the guidelines established by bodies like the Institutional Animal Care and Use Committee (IACUC) to ensure that their studies meet ethical standards.
  2. Logistical Challenges:
    • Housing Requirements: Different mouse strains have unique housing and environmental needs. Researchers must assess whether they can provide appropriate living conditions, including space, diet, and socialisation, to ensure the health and well-being of the animals.
    • Budget and Resources: Financial constraints can significantly impact the choice of animal models and the scope of research. It is essential to consider costs associated with purchasing, breeding, housing, and maintaining mouse colonies, as well as any specialised equipment or facilities required.

Tips:

To effectively navigate ethical and practical constraints, researchers should take a proactive and structured approach:

  • Adopt the 3 Rs of Animal Use:
    • Replacement: Consider alternatives to animal models where possible. This can include in vitro studies, computer modelling, or the use of lower organisms that do not require the same ethical considerations.
    • Reduction: Employ strategies to minimise the number of animals used in research. This could involve using statistical methods to ensure that the fewest number of animals necessary for statistically valid results are used.
    • Refinement: Implement practices that enhance animal welfare and minimise suffering. This can include optimising experimental procedures, improving housing conditions, and providing appropriate veterinary care.
  • Select Appropriate Models: Choose mouse models that align with ethical guidelines and can be supported by available resources. Consider models that are known for their robustness, ease of handling, and compatibility with existing protocols.
  • Plan Logistically: Conduct a thorough assessment of logistical factors before initiating a study. This includes ensuring that adequate facilities and resources are available and planning for potential challenges related to housing and breeding.
  • Engage with Ethical Review Boards: Actively involve ethical review boards or committees early in the research design process. They can provide valuable insights into ethical considerations and help researchers design studies that adhere to established guidelines.

By being mindful of ethical and practical constraints and implementing the 3 Rs, researchers can enhance the integrity of their studies and ensure that their work contributes positively to the scientific community while respecting the welfare of animal subjects.

References:

Beery AK. Inclusion of females does not increase variability in rodent research studies. Curr Opin Behav Sci. 2018 Oct;23:143-149. doi: 10.1016/j.cobeha.2018.06.016. Epub 2018 Aug 2. PMID: 30560152; PMCID: PMC6294461.

Chen X, Oppenheim JJ, Howard OM. BALB/c mice have more CD4+CD25+ T regulatory cells and show greater susceptibility to suppression of their CD4+CD25- responder T cells than C57BL/6 mice. J Leukoc Biol. 2005;78(1):114-121. doi:10.1189/jlb.0604341

Coleman, D.L. Obese and diabetes: Two mutant genes causing diabetes-obesity syndromes in mice. Diabetologia 14, 141–148 (1978). https://doi.org/10.1007/BF00429772

Michael F. W. Festing, Evidence Should Trump Intuition by Preferring Inbred Strains to Outbred Stocks in Preclinical Research, ILAR Journal, Volume 55, Issue 3, 2014, Pages 399–404, https://doi.org/10.1093/ilar/ilu036

Sarah F. Rosen et al. Olfactory exposure to late-pregnant and lactating mice causes stress-induced analgesia in male mice.Sci. Adv.8,eabi9366(2022).DOI:10.1126/sciadv.abi9366

Tuttle, A.H., Philip, V.M., Chesler, E.J. et al. Comparing phenotypic variation between inbred and outbred mice. Nat Methods 15, 994–996 (2018). https://doi.org/10.1038/s41592-018-0224-7

Trunova, G.V., Makarova, O.V., Diatroptov, M.E. et al. Morphofunctional Characteristic of the Immune System in BALB/c and C57Bl/6 Mice. Bull Exp Biol Med 151, 99–102 (2011). https://doi.org/10.1007/s10517-011-1268-1

Yoshiki, A., Ballard, G. & Perez, A.V. Genetic quality: a complex issue for experimental study reproducibility. Transgenic Res 31, 413–430 (2022). https://doi.org/10.1007/s11248-022-00314-w

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