1 Introduction

Genetically modified organisms (GMOs) have revolutionized the field of biomedical research by providing powerful tools to model human diseases, understand gene functions, and develop new therapeutic strategies. GMOs are organisms whose genetic material has been altered using genetic engineering techniques to introduce, remove, or modify specific genes. This technology has enabled researchers to create animal models that closely mimic human diseases, thereby facilitating the study of disease mechanisms and the development of potential treatments (Zhai et al., 2022).

The use of GMO animals in biomedical research is of paramount importance. Traditional animal models, such as mice, have been instrumental in advancing our understanding of various diseases. However, they often fall short in replicating the complexity of human conditions. For instance, genetically modified pigs have emerged as valuable models due to their physiological and anatomical similarities to humans, making them particularly useful for studying cardiovascular diseases, cancers, diabetes, and neurodegenerative disorders (Perleberg et al., 2018). These models not only enhance our understanding of disease pathology but also improve the translational potential of preclinical findings, thereby bridging the gap between basic research and clinical application.

The objectives of this study are to provide a comprehensive overview of the application of GMO animals in biomedical research and to assess the biosafety concerns associated with their use. We will explore the various genetic engineering techniques employed to create these models, highlight their contributions to medical science, and discuss the potential risks and ethical considerations. By examining the current state of GMO animal research and its future prospects, this study aims to underscore the significance of these models in advancing medical science while ensuring their safe and ethical use.

2 Development of GMO Animals for Biomedical Research

2.1 Techniques used in the creation of GMO animals

The development of genetically modified organisms (GMO) in biomedical research has been significantly advanced by various gene-editing techniques. Among these, CRISPR-Cas9 has emerged as a dominant tool due to its precision, efficiency, and versatility. This system allows for targeted modifications in the genome, enabling the creation of knockout and knockin models in various species, including pigs and chickens (Figure 1) (Tu et al., 2022; Park, 2023). Other techniques such as transgenesis, which involves the insertion of foreign genes into an organism, and gene knockout methods, which disable specific genes, are also widely used. Transgenesis can be achieved through methods like DNA microinjection, retroviral vectors, and embryonic stem cell manipulation. Additionally, older technologies like zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are still in use, although they have been largely supplanted by CRISPR-Cas9 due to its simplicity and effectiveness (Sundhari et al., 2019).

Figure 1 Genome/gene-editing nucleases (Adopted from Tu et al., 2022) Image caption: (A) Illustration of a pair of functional ZFNs bound to DNA. An N-terminal domain is shown to aid in the folding of zinc finger domains. Each tri-ZF is fused with the nuclease domain of FokI (FN) via a peptide linker. The recognition sites of each pair of ZFNs are organized in a tail-to-tail orientation to perform effective double-strand cutting activity. (B-1) A model consisting of a pair of TALENs in a head-to head orientation is shown. An N-terminal domain is also needed to facilitate the folding of the 34 AA repeat domains. A C-terminal domain containing an NLS is essential for enzyme activity. (B-2) The engineered TALE can be used as a sequence-specific DNA binding domain to carry a transcriptional regulator, DNA-modifying enzyme, or histone modification enzyme to the DNA region of interest. The domain organization of SpCas9 (C-a) and a schematic diagram of wild-type SpCas9 associated with a sg-RNA (C-b) are shown. (C-c) The noncomplementary strand is cut by the RuvC nuclease domain, and this nuclease activity is blocked in the D10A mutant. (C-e) The complementary strand is digested by the HNH nuclease domain, and this nuclease activity is blocked in the H840A mutant. (C-f) Both nuclease activities of SpCas9 are lost in the D10A/ H840A double mutant, which is referred to as dead Cas9 (dCas9). (C-d) The D10A mutant, also known as Cas9 nickase (nCas9), is engineered as a C to T nucleotide editor by linking a cytidine deaminase, APOBEC1, to its N-terminus, and the switching probability can be increased by the fusion of a uracil glycosylase inhibitor (UGI) to the C-terminus of nCas9. (C-g) Similar to TALE, dCas9 can be guided by a sgRNA as a sequence-specific DNA-binding roboprotein. Transcriptional regulators, DNA-modifying enzymes, or histone-modifying enzymes can be fused to either or both of the N- and C-termini (Adopted from Tu et al., 2022)

The study of Tu et al. (2022) illustrates various genome and gene-editing tools, specifically Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and CRISPR-Cas9 systems. ZFNs and TALENs rely on engineered proteins to target specific DNA sequences, leading to precise double-strand breaks, which are crucial for gene editing. The CRISPR-Cas9 system, depicted in multiple configurations, highlights the versatility of Cas9, which can be engineered to perform single-strand nicks, double-strand breaks, or even to recruit other molecules for targeted gene regulation without cutting DNA. These tools revolutionize genetic manipulation, enabling targeted and efficient modifications.

2.2 Key milestones in the development of GMO animals for research purposes

Several key milestones have marked the development of GMO animals for biomedical research. The initial use of chemical and physical mutagenesis methods in the 1980s laid the groundwork for genetic modifications, although these methods lacked precision. The advent of CRISPR-Cas9 revolutionized the field by enabling precise genome editing, which has been used to create various animal models, including pigs with specific gene knockouts for studying human diseases (Wu et al., 2017). Another significant milestone was the development of transgenic animals that ubiquitously express Cas9, facilitating in vivo genome editing without the need for germline modifications (Rieblinger et al., 2021). Additionally, the generation of gene-modified monkeys using CRISPR-Cas9 has provided valuable models for studying complex human diseases (Niu et al., 2014). These advancements have not only improved our understanding of genetic functions but also enhanced the development of therapeutic strategies.

2.3 Ethical considerations in the development and use of GMO animals

The development and use of GMO animals in biomedical research raise several ethical considerations. One major concern is animal welfare, as genetic modifications can lead to unintended health issues and suffering in the modified animals (Asfaw and Assefa, 2019). Ethical guidelines and regulations are essential to ensure that the use of GMO animals is justified and that their welfare is prioritized. Another ethical issue is the potential environmental impact if genetically modified animals were to escape into the wild, which could disrupt ecosystems. Additionally, there are concerns about the long-term effects and unforeseen consequences of genetic modifications, which necessitate thorough risk assessments and regulatory oversight. The ethical debate also extends to the moral implications of creating and using animals for research purposes, balancing scientific progress with humane treatment. These considerations underscore the importance of ethical frameworks and responsible practices in the field of genetic engineering.

3 Applications of GMO Animals in Biomedical Research

3.1 Use of GMO animals in understanding disease mechanisms

Genetically modified organisms (GMOs) have become indispensable tools in biomedical research, particularly in understanding the mechanisms of various diseases. For instance, genetically modified pigs have been developed to model human diseases such as cardiovascular diseases, cancers, diabetes mellitus, Alzheimer's disease, cystic fibrosis, and Duchenne muscular dystrophy. These porcine models are highly valuable due to their physiological and anatomical similarities to humans, which make them more suitable than traditional rodent models for translational research (Perleberg et al., 2018). Additionally, various animal models, including murine, primate, and aquatic species, have been utilized to study neurological, behavioral, cardiovascular, and oncological disorders, thereby advancing our understanding of these conditions and aiding in the development of new therapeutic approaches.

3.2 GMO animals as models for drug discovery and development

GMO animals play a crucial role in drug discovery and development by serving as models for preclinical testing. Regulatory agencies now require preclinical trial data from non-rodent species due to the limitations of rodent models in translating basic research into clinical applications. Genetically modified pigs, for example, are increasingly used in preclinical studies to test the efficacy and safety of new drugs, given their closer resemblance to human physiology and pathophysiology. Furthermore, the use of various animal models, including primates and rodents, has been instrumental in studying the pathology of diseases such as the 2019 Coronavirus, thereby facilitating the development of therapeutic protocols (Figure 2) (Domínguez-Oliva et al., 2023).

Figure 2 Classification of various animal models. The animals used in science can be divided into five broad types (Adopted from Domínguez-Oliva et al., 2023) Image caption: (a) The main ones are models in which animals are induced to present a pathology similar to one that affects humans or other animals by administering drugs or other biologicals, inflicting injuries, or subjecting them to stress or other environmental conditions. In contrast, models based on spontaneous changes (b) include animals where the normal course of their life predisposes them to develop a specific disease. (c) Genetically-modified test subjects are animals with knockin or knockout genes or proteins. In contrast to using healthy animals (e), negative models (d) employ individuals that are not susceptible to certain diseases but serve to evaluate susceptibility to a specific pathology. TBI: traumatic brain injury (Adopted from Domínguez-Oliva et al., 2023)

The study of Domínguez-Oliva et al. (2023) categorizes animal models into five types used in scientific research. These include induced disease models, where animals are given treatments or subjected to conditions that mimic human diseases, and spontaneous models, where natural predispositions lead to disease. Genetically modified models involve animals with specific genes altered to study particular functions or diseases. Negative models consist of animals resistant to certain diseases, useful for understanding disease susceptibility. Lastly, healthy animals serve as controls in experiments to provide baseline data or to study normal biological processes. Each model serves a distinct purpose in advancing biomedical research.

3.3 Role of GMO animals in regenerative medicine and gene therapy research

In the field of regenerative medicine and gene therapy, GMO animals are pivotal for assessing the safety and efficacy of new treatments. For example, Institutional Biosafety Committees (IBCs) in Australia have been actively involved in reviewing and advising on clinical trials involving viral vector gene therapies and gene-modified viruses. These trials include the use of adeno-associated viral vectors for hemophilia, oncolytic GM human HSV-1 for melanoma, and CRISPR Cas-9 engineered T-cells for B cell malignancies (O'Sullivan et al., 2020). Additionally, machine learning approaches are being integrated into stem cell research to assess biosafety and bioefficacy, addressing concerns such as tumorigenicity in stem cell therapy (Zaman et al., 2021). These advancements highlight the critical role of GMO animals in pioneering new treatments and ensuring their safety for clinical application.

4 Case Study: The Role of GMO Mice in Alzheimer’s Disease Research

4.1 Overview of alzheimer’s disease and the need for effective research models

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the most common cause of dementia, affecting millions of people worldwide. It is characterized by the accumulation of amyloid-beta plaques, neurofibrillary tangles, synaptic dysfunction, and neuronal death (Figure 3) (Sanchez-Varo et al., 2022). Despite extensive research, there is currently no cure for AD, and the available treatments only offer symptomatic relief. Therefore, there is a critical need for effective research models that can accurately mimic the human pathology of AD to facilitate the development of therapeutic interventions (Esquerda-Canals et al., 2017).

Figure 3 Amyloid plaque types in APP-based models. Neuritic plaques exhibit a fibrillar core encircled by a ring of oligomeric Aβ, and are surrounded by swollen neuronal projections, named dystrophic neurites. Conversely, diffuse plaques lack the fibrillar nucleus and do not display aberrant neuropile around them. Cerebral amyloid angiopathy (CAA) consists in vascular deposits of amyloid fibrils that accumulate within wall vessels of the brain. Parenchymal deposits are mainly formed by Aβ42, while vascular deposits are composed of Aβ40 (Adopted from Sanchez-Varo et al., 2022)

The study of Sanchez-Varo et al. (2022) illustrates three types of amyloid plaques observed in APP-based models of Alzheimer's disease: neuritic plaques, diffuse plaques, and vascular deposits. Neuritic plaques have a fibrillar core surrounded by dystrophic neurites, primarily composed of Aβ42. Diffuse plaques, which lack a fibrillar core and associated dystrophic neurites, also consist mainly of Aβ42. Vascular deposits, associated with cerebral amyloid angiopathy (CAA), consist of amyloid fibrils accumulating within the walls of brain vessels, predominantly composed of Aβ40. These different plaque types reflect the diverse pathological features in Alzheimer’s disease, contributing to varying effects on brain function.

4.2 Development of GMO mice to mimic human alzheimer’s pathology

Genetically modified organisms (GMO) mice have been instrumental in AD research. The development of transgenic mouse models began with the identification of heritable mutations in genes such as amyloid precursor protein (APP) and presenilins (PSEN1 and PSEN2), which are linked to familial AD (FAD) (Scearce-Levie et al., 2020). These models overexpress human genes carrying mutations associated with early-onset AD, leading to the formation of amyloid plaques and other AD-related pathologies (Carmo and Cuello, 2013). More recent models have incorporated multiple genetic risk factors and humanized sequences to better replicate the complexity of the disease. These advancements have allowed researchers to study the disease's progression and test potential therapeutic strategies in a controlled environment (Sasaguri et al., 2017).

4.3 Contributions of these models to our understanding of the disease and therapeutic advancements

GMO mice have significantly contributed to our understanding of AD by elucidating the mechanisms underlying amyloid plaque formation, tau pathology, synaptic dysfunction, and neuroinflammation (Drummond and Wisniewski, 2017). These models have been used to identify and validate therapeutic targets, leading to the development of potential treatments that can be tested in preclinical studies (Croft and Noble, 2018). For instance, transgenic mice have been crucial in the discovery of biomarkers and the evaluation of pharmacokinetic and pharmacodynamic properties of new drugs (LaFerla and Green, 2012). Although no model perfectly replicates human AD, the insights gained from these studies have been invaluable in advancing the field and bringing preclinical research closer to clinical applications.

5 Biosafety Concerns Associated with GMO Animals

5.1 Potential risks associated with the use of GMO animals

The use of genetically modified organisms (GMOs) in biomedical research, including GMO animals, presents several potential risks. One significant concern is genetic drift, where unintended genetic changes occur over generations, potentially leading to unforeseen consequences. Additionally, unintended gene expression can result from the insertion of foreign genes, which may disrupt normal gene function and lead to unexpected phenotypic traits (Andersson et al., 2012). These risks necessitate thorough molecular characterization and phenotypic comparisons between GMO animals and their non-GMO counterparts to ensure safety and stability.

5.2 Environmental risks and containment strategies

Environmental risks associated with GMO animals include the potential for these organisms to escape containment and interact with wild populations, leading to ecological imbalances. The introduction of GMO animals into the environment could result in the transfer of modified genes to wild species, potentially disrupting local ecosystems. To mitigate these risks, stringent containment strategies are essential. These strategies include physical barriers, such as secure enclosures, and biological containment measures, such as genetic modifications that prevent reproduction outside controlled environments (Beeckman and Rüdelsheim, 2020). Regulatory frameworks, such as those outlined by the European Food Safety Authority (EFSA), emphasize the importance of environmental risk assessments (ERAs) to evaluate and manage these potential impacts (Eckerstorfer et al., 2023).

5.3 Ethical concerns related to animal welfare and genetic integrity

The ethical concerns surrounding the use of GMO animals in research primarily revolve around animal welfare and the preservation of genetic integrity. Genetic modifications can lead to unintended health issues in animals, raising questions about their well-being and the ethical implications of using such animals in research. Comparative analyses of health and physiological parameters between GMO and traditionally bred animals are crucial to ensure that the modifications do not adversely affect the animals' health. Additionally, there are broader ethical debates about the manipulation of genetic material and the potential long-term consequences for biodiversity and genetic integrity (Kamle and Ali, 2013). These concerns highlight the need for robust ethical guidelines and oversight to balance scientific advancement with animal welfare and environmental stewardship (Ghimire et al., 2023).

6 Regulatory Framework for GMO Animals in Biomedical Research

6.1 Overview of international and national regulations governing the use of GMO animals

The regulatory landscape for genetically modified organisms (GMOs), including animals, is complex and varies significantly across different countries and international bodies. Internationally, the Cartagena Protocol on Biosafety is a key instrument that provides a framework for the safe transfer, handling, and use of GMOs, with a focus on protecting biodiversity and human health (Komen, 2012). At the national level, regulations can differ widely. For instance, the European Union (EU) has a stringent regulatory framework that includes directives such as Dir 90/220/EEC, which was one of the first to address the deliberate release of GMOs into the environment (Eriksson, 2018). In contrast, regulatory approaches in countries like the United States and Brazil may focus more on the product rather than the process, leading to different regulatory triggers and assessments (Eckerstorfer et al., 2019).

6.2 Guidelines for biosafety assessment and risk management

Biosafety assessment and risk management are critical components of the regulatory framework for GMO animals. These assessments typically involve a comprehensive evaluation of potential risks to human health, animal welfare, and the environment. The European Food Safety Authority (EFSA) provides detailed guidance on the risk assessment of GM animals, which includes molecular characterization, toxicological assessment, allergenicity, and nutritional evaluation (Andersson et al., 2012). This guidance emphasizes a comparative approach, assessing GM animals against their non-GM counterparts to identify any significant differences that could pose risks. Additionally, the principles of risk assessment and management are universally applied, focusing on identifying hazards, assessing exposure, and implementing measures to mitigate identified risks (Beeckman and Rüdelsheim, 2020).

6.3 Case examples of regulatory compliance in different countries

Different countries have adopted various strategies to ensure regulatory compliance for GMO animals. In the EU, the regulatory framework has evolved to include a case-by-case assessment approach, ensuring that each GMO is evaluated based on its specific characteristics and potential risks. For example, the EU's mandatory environmental risk assessment (ERA) for GMOs ensures a high level of protection before any GMO is authorized for environmental release or marketing (Eckerstorfer et al., 2023). In contrast, countries like Australia and New Zealand have developed their own risk assessment frameworks, which include specific guidelines for evaluating the potential adverse effects of GMOs, including those producing new regulatory-RNA molecules (Heinemann et al., 2013). These frameworks are designed to be flexible, allowing for updates and improvements as new scientific knowledge and technologies emerge.

7 Biosafety Assessment Strategies

7.1 Methodologies for assessing the biosafety of GMO animals

The assessment of biosafety in genetically modified organisms (GMOs), including animals, involves a comprehensive evaluation of potential risks to human health, animal health, and the environment. Methodologies for biosafety assessment typically include a combination of in vitro and in vivo testing, computational modeling, and regulatory frameworks. For instance, the European Food Safety Authority (EFSA) emphasizes a case-by-case approach for environmental risk assessment (ERA) of GMOs, ensuring that each GMO is evaluated based on its specific characteristics and potential risks (Eckerstorfer et al., 2023). Additionally, the integration of alternative methods, such as in vitro-based risk evaluation processes, is gaining traction to reduce reliance on animal testing (Leist et al., 2012).

7.2 In vivo and in vitro testing approaches

In vivo testing involves the use of live animals to study the effects of GMOs, which remains a critical tool for understanding disease mechanisms and developing treatments (Alderman et al., 2018). However, ethical concerns and the need for more rapid and cost-effective methods have led to the development of in vitro testing approaches. These include the use of 2D and 3D cell cultures, organotypic microtissues, and high-throughput omics data to predict adverse outcomes (Jagiello and Ciura, 2022). In vitro to in vivo extrapolation (IVIVE) models are also being developed to bridge the gap between laboratory findings and real-world biological responses, enhancing the predictive power of in vitro assays.

7.3 Risk assessment models and decision-making frameworks

Risk assessment models for GMOs incorporate various factors, including the potential for environmental release, human exposure, and unintended effects. The Organisation for Economic Co-operation and Development (OECD) has developed the concept of systemic risks, which includes socio-economic aspects alongside traditional health and environmental risks (Meyer, 2011). Regulatory frameworks, such as those in the EU and the USA, often differ in their approach to risk assessment, with some focusing on the process of genetic modification and others on the characteristics of the resulting product (Eckerstorfer et al., 2019). Decision-making frameworks must balance scientific evidence with public concerns, regulatory requirements, and the potential benefits of GMOs. For example, early stakeholder engagement and transparent communication are crucial for fostering public acceptance and avoiding regulatory bottlenecks (Pillai and Raybould, 2023).

8 Challenges in Biosafety Assessment and Risk Management

8.1 Difficulties in predicting long-term effects of genetic modifications

One of the primary challenges in the biosafety assessment of genetically modified organisms (GMOs) is the difficulty in predicting long-term effects. Genetic modifications can have unforeseen consequences that may not be immediately apparent. For instance, the introduction of new regulatory-RNA molecules through genetic engineering can create biosafety risks that are not fully understood or anticipated (Heinemann et al., 2013). Additionally, the long-term environmental impacts of GMOs, such as their interaction with non-target species and ecosystems, remain a significant concern (Schiemann et al., 2019). The complexity of these interactions makes it challenging to develop comprehensive risk assessment models that can accurately predict long-term outcomes.

8.2 Gaps in current biosafety assessment protocols

Current biosafety assessment protocols often fall short in addressing the multifaceted risks associated with GMOs. For example, the existing frameworks primarily focus on immediate environmental and health risks, often neglecting socio-economic and ethical considerations (Meyer, 2011). The European Food Safety Authority (EFSA) has been criticized for its lack of specific guidance on the environmental risk assessment (ERA) of genome-edited plants, which highlights the need for more detailed and case-specific protocols. Moreover, the regulatory frameworks in different countries vary significantly, leading to inconsistencies in risk assessment and management practices (Eckerstorfer et al., 2019). This lack of harmonization complicates international trade and the global acceptance of GMOs.

8.3 Strategies to overcome these challenges, including new technologies and approaches

To address these challenges, several strategies and new technologies are being proposed. One approach is to expand the scope of risk assessments to include more holistic concepts, such as the Organisation for Economic Co-operation and Development (OECD) concept of systemic risks, which incorporates socio-economic aspects. Another strategy is to develop more specific and detailed guidance for the ERA of genome-edited plants, as suggested by the European Commission (Eckerstorfer et al., 2023).

Advancements in molecular biology, such as real-time PCR and other bioanalytical methods, offer new tools for the sensitive detection and traceability of GMOs (Kamle and Ali, 2013). These technologies can help in monitoring the expression of transgenes and their potential impacts more accurately. Additionally, international cooperation and the establishment of public registries for biotechnology products can enhance transparency and facilitate the harmonization of regulatory frameworks.

9 Future Directions in the Application and Biosafety of GMO Animals

9.1 Emerging trends in GMO animal research and potential new applications

The field of genetically modified organisms (GMO) in animal research is rapidly evolving, with several emerging trends and potential new applications. One significant trend is the use of genome editing techniques, such as CRISPR-Cas9, to create precise genetic modifications. These techniques are being applied to develop animal models that can better mimic human diseases, thereby enhancing biomedical research and drug development (Pillai and Raybould, 2023). Additionally, there is growing interest in using GMO animals for agricultural purposes, such as improving disease resistance and enhancing productivity in livestock (Komen et al., 2020). Another promising application is the development of transgenic animals that can produce therapeutic proteins or other biologically active substances, which can be used in the treatment of various diseases.

9.2 Innovations in biosafety assessment technologies

Innovations in biosafety assessment technologies are crucial to ensure the safe application of GMO animals. Recent advancements include the development of next-generation biocontainment systems that utilize synthetic biology techniques to prevent the unintended release of genetically modified organisms into the environment (Lee et al., 2018). These systems involve genetic circuit engineering, genome editing, and gene expression regulation to create robust containment strategies. Additionally, there is a push towards more comprehensive risk assessment frameworks that incorporate not only environmental and health risks but also socio-economic and ethical considerations Meyer, 2011; Heinemann et al., 2013). The use of advanced analytical tools and computational models to predict and assess the potential impacts of GMO animals is also gaining traction, providing more accurate and reliable biosafety evaluations (Beeckman and Rüdelsheim, 2020).

9.3 The future of regulatory oversight in the context of rapidly advancing genetic technologies

As genetic technologies continue to advance rapidly, regulatory oversight must evolve to keep pace with these developments. One of the key challenges is the harmonization of regulatory frameworks across different countries to ensure consistent and effective biosafety standards (Eckerstorfer et al., 2023). There is a growing recognition of the need for a more flexible and adaptive regulatory approach that can accommodate the unique characteristics of new genetic modification techniques (Schiemann et al., 2019). This includes the development of specific guidelines for the environmental risk assessment of genome-edited organisms and the establishment of international public registries to enhance transparency and traceability of GMO products (Eckerstorfer et al., 2019). Furthermore, early stakeholder engagement and public communication are essential to foster acceptance and trust in GMO technologies, which can help mitigate regulatory bottlenecks and facilitate the safe and responsible use of GMO animals in biomedical research and other applications (Gronvall, 2017).

10 Concluding Remarks

In this study, we have explored the application and biosafety assessment of genetically modified (GMO) animals in biomedical research, with a particular focus on genetically modified pigs. The key findings from the study highlight the significant advancements in genetic engineering techniques that have enabled the development of transgenic animal models, particularly pigs, which are highly relevant for studying human diseases and for applications such as xenotransplantation. These models have been instrumental in understanding disease mechanisms and in the development of new therapeutic strategies.

Despite the promising applications, the biosafety assessment of GMO animals remains a critical concern. Current methodologies for risk assessment, including molecular characterization and biocontainment strategies, have shown limitations. For instance, traditional 90-day tests for evaluating chronic toxicity in GMO diets are insufficient, and there is a need for more comprehensive and prolonged studies. Additionally, innovative biocontainment strategies, such as the use of synthetic amino acids to create dependency in genetically modified organisms, have shown potential but require further refinement to ensure robustness against evolutionary escape.

To improve the application and biosafety assessment of GMO animals, it is recommended that regulatory frameworks be updated to include more holistic and comprehensive risk assessment methodologies. This includes the integration of socio-economic and environmental aspects into the biosafety evaluation process. Moreover, the development and implementation of advanced genetic engineering techniques, such as LIFE-Seq for precise molecular characterization, should be prioritized to enhance the accuracy and reliability of biosafety assessments.

Interdisciplinary collaboration is essential to ensure the safe and ethical use of GMO animals in biomedical research. Scientists, regulatory authorities, ethicists, and other stakeholders must work together to address the complex challenges associated with the use of genetically modified animals. This collaborative approach will help to develop robust biosafety protocols, promote transparency, and foster public trust in the use of GMO animals for scientific and medical advancements.

Acknowledgments

EcoEvo Publisher thanks the anonymous reviewers for their insightful comments and suggestions that improved the manuscript.

Conflict of Interest Disclosure

The author affirms that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

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