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The Advantage and Application of Genetically Humanized Mouse Models for Biomedical Research 1 TACONIC BIOSCIENCES INTRODUCTION The use of genetically engineered mice in experimental medical research has led to significant advances in our understanding of human health and disease. From the development of transgenic and gene targeting methods to recent innovations in gene-editing technologies, manipulation of the mouse genome has become increasingly sophisticated. Although there is extensive genetic similarity between humans and mice, it is now well recognized that even very subtle sequence differences between the two species can have important functional consequences for the respective individual gene functions1. In addition, a number of genes have been identified in humans that do not have orthologous mouse counterparts2. This divergence limits the utility of mouse models in predicting gene function and as a reliable tool for preclinical research. As a result, there is growing interest in the generation of genetically engineered mice that express an orthologous human gene or even entire human genomic loci1,3. These genetically “humanized” mice have the potential to provide more reliable in vivo data concerning human gene function in normal physiology and disease. The purpose of this white paper is to discuss the various technologies involved in the generation of genetically humanized mice and the applications of these models in biomedical research. HUMANIZATION OF THE MOUSE GENOME: FROM RANDOM TRANSGENESIS TO TARGETED REPLACEMENT Transgenic Technology The development of transgenic mouse technology in the 1970s and 1980s remains a major milestone in the history of molecular biology and mouse genetics4. Now routine in many laboratories, this technology was made possible through important discoveries in the hormonal control of reproduction, advancements in the manipulation of mouse embryos and the use of recombinant DNA technology5. For the purposes of this white paper, the term “transgenic” refers to mice carrying exogenous DNA that has integrated within the genome and is expressed “in trans” (ie. not within its native genetic locus). The first strains of transgenic mice were generated through viral infection of preimplantation mouse embryos6. While this method led to successful germline transmission of the foreign DNA, active repression of the integrated genetic material by host factors resulted in a high degree of mosaicism. A major breakthrough occurred 2 TACONIC BIOSCIENCES in the early 1980s with the development of a gene transfer method in which DNA could be directly injected into the pronuclei of zygote-stage embryos7. Still extensively used today, the pronuclear microinjection procedure allows for essentially any DNA sequence to be introduced into the mouse genome with reasonably high efficiency. The initial strains of transgenic mice produced by this route carried genes of viral7,8, rat9 and rabbit10,11 origin. The first report of a humanized transgenic mouse appeared in 1983 for mice carrying an actively expressed human growth hormone gene9. Since these early reports, thousands of transgenic mice have been generated and characterized to define the function of human genes in normal development and disease pathogenesis. Transgenic technology has been widely used as a method to overexpress human genes of interest using ubiquitous or tissue-specific promoters. However, limitations associated with transgenesis have the potential to complicate the interpretation of data obtained from these studies. For instance, the integration site can affect the level and spatial pattern of transgene expression; a phenomenon referred to as position effects. In addition, multiple copies of the transgene are often incorporated into the chromosome as a head-to-tail concatemer. While the reasons for this remain unclear, concatemers can be unstable resulting in deletion of one or more copies of the transgene. In some cases, transgene integration can occur within an endogenous gene disrupting its function and causing phenotypic changes that are unrelated to the gene of interest. Since the probability of obtaining different founders with the same integration site is low, molecular and phenotypic characterization of multiple founders is required to accurately determine the functional role of the transgene12. Gene Targeting A more precise method for manipulating the mouse genome, termed gene targeting, was developed in the late 1980s based on the work of Mario Capecchi13, Martin Evans14 and Oliver Smithies 15. This powerful technique was made possible through the use of homologous recombination (HR) and newly created techniques for the isolation and culture of mouse embryonic stem cells (ES cells). Homologous recombination functions in the exchange of similar or identical nucleotides and is critical for the DNA repair process. To modify a specific locus within the mouse genome, a targeting construct is engineered with the sequence to be inserted flanked by regions of homology to the desired integration site. The targeting construct is then transferred into ES cells via electroporation. Since HR occurs at a low frequency (10-2 10-3 of integrations are HR events), drug selection markers are incorporated into the targeting construct to allow for enrichment of recombinant ES cell clones16. Isolated clones are then injected into the fluid-filled blastocoel cavity of early embryos to generate chimeric blastocysts that are surgically transferred into pseudopregnant females. Germ-line transmission of the targeted allele is then achieved by breeding chimeras with wild type mice resulting in the generation of mice heterozygous for the targeted mutation. A final round of breeding is then carried out between heterozygotes to produce the desired homozygous animals. Due to the immense contributions of gene targeting to the scientific field, it is not surprising that the three scientists who pioneered the method, were awarded the 2007 Nobel Prize for Physiology or Medicine17. Gene targeting technology allows for the replacement of the homologous mouse gene with the human counterpart or insertion of the human gene into an unrelated but permissive site within the genome. These ‘safe harbor’ sites within the mouse genome permit a predictable pattern of gene expression without disrupting the function of 3 TACONIC BIOSCIENCES essential endogenous genes18. The most common permissive regions that have been used to generate knock-in mice include the ROSA26 and collagen (Col1a1) loci which direct constitutive and ubiquitous expression of transgenes19,20. Tissue specificity of expression of human transgenes can be achieved using this approach by using a ubiquitous promoter separated from the transgene by a loxP-flanked stop cassette and crossing this model to a mouse line expressing Cre recombinase in a tissue-specific manner21. HUMAN DNA IN MICE: TYPES OF DNA MOLECULES FOR HUMANIZATION The first generation of genetically engineered mice carrying and expressing human genes was relatively simple in design. Typically, a human gene of interest in the form of cDNA was placed under control of a small heterologous promoter to drive ubiquitous or tissue-specific transgene expression and inserted randomly into the genome. Alternatively, cDNA containing the human transgene and small promoter fragment can be targeted to a specific location within the genome. Since cDNA constructs lack the non-coding and regulatory elements found in genomic DNA, and are often under the control of a heterologous promoter, the level and pattern of transgene expression may not be reflective of what occurs under normal physiological conditions. Hence, the development of bacterial artificial chromosomes (BACs), which can accommodate large fragments of DNA has been crucial for the generation of improved transgenic mouse models22. The capacity of these vectors to carry large portions of DNA permits the inclusion of key regulatory domains that are essential for optimal levels of transgene expression and eliminates the impact of position effects when inserted randomly into the genome. Furthermore, large genomic vectors allow for the expression of splice variants, which can contribute significantly to the functional activity of the human gene. One of the major drawbacks with the random humanization approach is the continued presence and expression of the endogenous mouse gene. Since this can complicate the analysis of resulting phenotypes, more complex models, whereby the humanized transgenic mouse is bred onto a null background of the corresponding mouse gene, have been generated. This has been referred to as ‘knockout (KO) plus transgenic’ humanization23. In addition, subtle targeted mutations such as humanizing point mutations and small insertions and deletions can be introduced into the endogenous mouse gene. This strategy is particularly useful for the study of genetic variants associated with human diseases. For example, a recent study in Nature described the generation of a mouse strain harboring a SNP genetic variant strongly associated with Crohn’s disease that is useful for understanding the molecular pathogenesis of the disease24. Humanization can also be achieved with genomic fragments termed minigenes. These are compact versions of a gene containing only the necessary exons and regulatory elements required for functional protein production. The small size of minigenes makes them easier to manipulate compared to the full length version of the gene and they have been particularly useful for the study of alternative splicing in disease25. A more direct humanization approach involves targeted replacement of a portion of the mouse gene with small fragments of the human sequence (mouse-human chimeric gene) or replacement of the entire endogenous locus with the human ortholog23. While considerably more challenging from a technical standpoint, this strategy avoids 4 TACONIC BIOSCIENCES the laborious process of crossing transgenics onto a null background. For example, mice expressing a mouse-human chimeric p53 protein in which several exons of the mouse gene were replaced with the human sequence exhibit more accurate responses to DNA damaging agents and provide a useful tool for investigating the function of induced human p53 mutations in carcinogenesis26. Complete replacement of the mouse gene with the human counterpart can also be achieved. Although humanization of small genes has become relatively easy, the ability to replace large genes (>100kb) remains technically challenging but will become more routine with further advances in technology. Indeed, Taconic continues to play an active role in advancing this technology, and has become a world-leading expert in the generation of genetically humanized mouse models with large genomic replacements. APPLICATIONS OF GENETICALLY HUMANIZED MICE Genetically humanized mice have potential for use in a wide variety of research applications. Major areas in which these models have been successfully used are for the analysis of compound and biological efficacy and safety testing, novel therapeutic approaches, drug metabolism and disposition, and investigation into immune system development and function. Efficacy and Safety Testing of Therapeutic Compounds and Biologics Species differences in the interaction of therapeutic agents with a potential molecular target can limit the utility of wild type mice as a pre-clinical tool for efficacy and safety testing. To overcome this limitation, mice expressing the human version of a particular drug target can be generated. The generation of mPGES-1 knock-In mice provides an excellent example of the use of humanized mice for drug efficacy and safety application27. Microsomal prostaglandin E synthase-1 mediates the production of the major pro-inflammatory molecule PGE2. To test whether inhibition of mPGES-1 can block inflammation, a selective PGES-1 inhibitor, MF63, was tested in animal models of inflammation including mice. While MF63 strongly inhibited human mPGES-1 in human cells in vitro, the compound showed minimal activity against mouse mPGES-1. In collaboration with Taconic, a part of the mouse ptges gene was replaced with a human ptges minigene. As expected, treatment of mPGES-1 knockin mice with MF63 blocked PGE2 synthesis and reduced inflammatory responses in challenged mice. A novel humanized mouse model for the glucagon-like-peptide-1 receptor (GLP-1R) has also recently been reported28. GLP-1R mediates the effects of GLP-1 and plays an important role in regulating insulin secretion. Patients with type 2 diabetes often exhibit reduced levels of GLP-1 levels leading to extensive research into the identification of novel therapies targeting the GLP-1/GLP-1R pathway such as GLP-1 mimetics and GLPR1 agonists. Due to inter-species differences, the identification of small molecule ligands for GLPR1 may prove difficult. Therefore, in collaboration with Taconic, mice in which the mouse GLPR1 gene was replaced with the human counterpart were generated. These hGLPR-1R knockin mice allow for the purification of GLP-1R binding partners and the testing of novel therapies targeting human GLP-1R 5 TACONIC BIOSCIENCES A recent study in Nature Medicine highlights the power and utility of xenobiotic receptor humanized mice in the identification of regulatory pathways involved in mediating drug induced liver toxicity29. Tuberculosis (TB) is a global health problem that can be effectively controlled by combination treatment with rifampicin and isoniazid. However, a major limitation of these chemotherapies is the induction of liver injury and in some cases even liver failure. As a result, therapy is often altered or discontinued which can lead to relapse of the disease. Treatment of wild type mice with rifampicin and isoniazid fails to mimic the hepatoxicity that is observed in humans due to the weak effect of these drugs on the mouse pregnane X receptor (PXR). Therefore, Li et al. turned to the use of genetically engineered mice expressing the human version of PXR. Remarkably, PXR-humanized mice treated with rifampicin and isoniazid led to altered metabolic profiles and signs of severe liver damage similar to the toxicity that is characteristic of human TB patients under chemotherapy with the same agents. Further work revealed that co-treatment with these drugs caused accumulation of an endogenous hepatotoxin through a PXR-mediated metabolizing pathway. Thus, the use of humanized mice in this study offers novel insight into the mechanisms of drug toxicity and provides a basis for the development of novel strategies to predict, prevent and treat liver injury due to anti-TB therapy. Genetically Humanized Mouse Models of Drug Metabolism and Disposition Model systems that accurately predict the fate of a drug in humans are essential in pharmaceutical research. Due to significant species differences in the proteins utilized for drug metabolism and disposition, genetically humanized mouse models have emerged as an important tool for the study of human drug responses in an in vivo setting3. Humanized mouse models have been generated for different components of drug metabolism and disposition pathway. These include xenobiotic receptors, phase 1 and 2 metabolizing enzymes and drug transporters3. Advanced humanized models have also been generated in which individual genetic modifications have been combined to study the complex pathways involved in drug metabolism and disposition that occur in vivo. For example, knock-in mice carrying humanized versions of the xenobiotic receptors, PXR and CAR, under control of the endogenous mouse promoter have been independently generated and then crossed to generated double humanized mice30. These mice express PXR and CAR receptors in appropriate tissues and splice variants that have been described in humans. The generation of PXR and CAR humanized mice via knock-in techniques nicely illustrates the impact different humanization strategies can potentially have on the resulting phenotype and overall utility of a particular model. Mice carrying human versions of the PXR and CAR genes were previously generated using a knockout plus transgenic strategy. This involved transgenic expression of pure a cDNA for PXR or CAR under the control of a heterologous promoter followed by breeding onto a null background to ensure human-specific expression31–33. However, due to the use of sub-optimal promoters and a minimal cDNA construct, these animals failed to express the receptors in all tissues and lacked expression of human splice variants. Though this demonstrates the potential limitations of humanization strategies that combine 6 TACONIC BIOSCIENCES transgenic technology with gene knockout methods, it should be noted that this approach has been successfully applied to the generation of numerous humanized models and that the utility of these models is highly dependent upon the experimental question being addressed. One of the great promises of humanized mouse models for genes involved in drug metabolism and disposition is to enable quantitative predictions for drug responses in man. These results could then be used to select the most promising drug candidates, to define the starting doses before first test in man or determine if a clinical drug-drug interaction (DDI) study is warranted. For example, the utility of a PXR-CAR-CYP3A4 triple humanized mouse model to quantitatively predict PXR/CYP3A4-mediated clinical DDIs has been demonstrated34. Furthermore, a CYP3A4 humanized mouse model was successfully used to predict the hepatic clearance of CYP3A4 substrates in humans35. The use of genetically engineered mouse models for drug metabolism and disposition is a rapidly growing area of research. Readers interested in learning more about this field are directed to recent reviews providing excellent coverage of the utility and limitations of these models3,36,37. In response to the need for more reliable pre-clinical models for pharmaceutical research, Taconic offers the most complete collection of genetically humanized mice for the prediction of Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) profiles of therapeutic agents in humans. Distributed under the tADMET™ portfolio, these unique mouse models carry humanized modifications in multiple pathway components including xenobiotic receptors, phase 1 and phase 2 drug metabolizing enzymes and drug transporters. Humanization of these models includes knockout plus transgenic strategies as well as targeted replacement of the mouse locus with the corresponding human gene. As the components of tADMET™ are constantly evolving, a complete list of available models can be found on the Taconic website. A comprehensive white paper entitled ‘Challenges and Prospects in Predicting the ADME and Toxicity Characteristics in of Drugs in Humans’ can also be accessed through the Taconic website. Humanized Models to Study the Relevance of Human Gene Variants Humanized mice have also been utilized to examine the function of SNP variants as risk factors in alcohol use disorders38. A polymorphism in the OPRM1 gene, 188G, has previously been linked to excessive alcohol use. To directly establish a causal role for this variant, a humanized mouse line was generated in which exon 1 of the mouse OPRM1 gene was replaced with the corresponding human sequence. A second humanized line was generated using site-directed mutagenesis to introduce the 118G variant. Remarkably, an enhanced neurological response to alcohol was restricted to mice carrying the 118G allele indicating that the OPRM1 A118G variation is a likely genetic determent that modulates the response to alcohol. 7 TACONIC BIOSCIENCES Genetically Humanized Mice to Test Novel Therapeutic Approaches In Vivo Gene editing has recently emerged as an efficient method for introducing precise genetic alterations within the genome. One of the promising applications of gene editing technologies in human medicine is for the correction of disease-causing mutations. Proof of this concept was recently shown in which zinc finger nucleases (ZFNs) were employed to correct hemophilia B in a humanized mouse model of the disease. Hemophilia B is a clotting disorder characterized by low circulating levels of blood coagulation factor IX due to mutations in the F9 gene. To generate a humanized model of this disorder, a mutant human F9 minigene was knocked into the Rosa26 locus in collaboration with Taconic. These humanized mice were then bred onto an F9 null background to remove the endogenous mouse F9 gene. As expected, humanized F9 mutant mice led to significant reductions in circulating factor IX protein. However, introduction of a wild type F9 gene into the liver using viral-mediated ZFNs stabilized the levels of human factor IX and rescued the disease phenotype. This study highlights the utility of humanized mouse models in studying human disease and suggests that gene editing may be a possible strategy for the treatment of simple genetic disorders. Genetically Humanized Mice for the Immune System The immune system represents a complex network of cells, tissues and organs that collectively serve as a host defense mechanism against foreign agents. The specificity of the immune response and significant sequence divergence between mice and humans limits the use of wild type animals for the study of immune system development and function39. Thus, genetic engineering technologies that permit humanization of mouse genes play an important role in immunological research. HLA Humanized Mice The development of MHC class-I transgenic mice was one of the first applications of genetically humanized mice for the study of the immune system40. The major histocompatibility complex (MHC), referred to as human leukocyte antigen (HLA) in humans, is a group of cell surface molecules that process and present antigens derived from invading pathogens. The first generation of HLA humanized mice were relatively simple in design and ectopically expressed human HLA transgenes. Second generation models expressing human HLA and human CD8 chimeric transgenes improved recognition of mouse cells with the humanized MHC molecules with third generation models incorporating the expression of the HLA and CD8 transgenes onto an null background for the corresponding mouse MHC orthologs40. HLA humanized mice have been utilized for the generation of improved animal disease models, identification of novel epitopes from viruses and cancerous cells, and to study the efficacy of immunotherapies. In addition, breeding HLA transgenic mice into an immunodeficient background provides an appropriate thymic environment that allows for the study of human T-cell maturation and antigen-specific restriction41. Taconic distributes a number of transgenic HLA humanized mouse models, which express different serotypes including A1, A2.1, A11, A24, B7 and B44 and are available off-the-shelf. For more information, please refer to the Taconic website as well as a webinar entitled ‘HLA transgenic mice: development, validation and applications’. 8 TACONIC BIOSCIENCES T Cell Receptor Humanized Mice A number of humanized mouse models have also been generated for the T-cell receptor (TCR), a complex integral membrane molecule crucial for cell-mediated immunity42. The most complex model involved the use of YAC-based transgenic technology to individually generate human TCRα transgenic (hTRA-Tg) and TCRβ– transgenic (hTRB-Tg) mice43. These strains were then crossed to generated double humanized mice and further bred into a null background for the endogenous mouse c TCRα and TCRβ genes. Finally, the incorporation of an HLA transgene to facilitate increased production of CD8+ T-cells, led to a triple transgenic, double knockout mouse. This genetically highly complex model has great potential for use in the identification of T-cell receptors against human self-antigens such as tumor-associated antigens and human pathogens. Generation of Therapeutic Antibodies The use of humanized mice for the generation of fully humanized monoclonal antibodies has recently generated much excitement in the scientific community. First generation models were designed using a knockout plus transgenic approach in which randomly inserted human immunoglobulin (Ig) transgenes were expressed on a mouse Ig null background44. These models already represented a great advance in the field, but in some cases demonstrated a reduced antibody response to some antigens. This is due in part to the random insertion of the human transgenes within the mouse genome during transgenesis, and the associated effects as discussed earlier. To overcome these vagaries, a number of humanized mouse strains have been generated through total replacement of the mouse Ig repertoire with the corresponding human sequences45, all of which are reportedly capable of producing human-mouse/rat hybrid antibodies that can be rapidly converted to fully humanized molecules. The increasing number of platforms entering this area of research highlights the promise of humanized mice in the development of therapeutics for human disease. In fact, in some cases, these humanized antibodies have already been transitioned into the clinic. Humanized Mice for Enhanced Hematopoietic Reconstitution Engraftment of immunodeficient mice with human cells or tissues is commonly used to study the immune system and hematopoiesis41. For instance, transfer of human hematopoietic stem cells into immunodeficient strains of mice, such as the NOG mouse, results in multi-lineage differentiation and the development of multiple blood cell types. However, support of certain cell types, particularly myeloid and natural killer (NK) cells, is often limited due to a lack of interaction between mouse cytokines with the cognate human receptors46. To overcome this, a variety of genetically engineered humanized mice have been generated that express the human version of essential cytokines. These include transgenic or knock-in mice for expressing human thrombopoietin47, M-CSF48, GM-CSF and IL-349. Recently, mice expressing all 4 of these human cytokine genes on an immunodeficient background have been described50. This complex model, which contains 3 knock-in and 2 knockout alleles, supports the development of multiple cell types of the innate immune system including monocytes, macrophages, NK cells and has potential use in the study of immune cell formation, infectious disease and cancer. 9 TACONIC BIOSCIENCES For a more detailed discussion of cell and tissue humanized mice, please refer to our recent white paper entitled ‘Latest Advances in Cell and Tissue Humanized Mice’ which is available through the Taconic website. Other Applications Genetically humanized mice also have application to a variety of other areas of experimental medical research. These include but are not limited to infectious disease (hepatitis C and HIV research), aneuploidy (Down’s Syndrome), Huntington disease, cancer research and models available through Taconic for cardiovascular disease (APOE3 and APOE4 humanized mice) and neurology (humanized Tau mouse). GENETIC HUMANIZATION WITH TACONIC In addition to offering a large number of humanized mouse strains “off the shelf,” Taconic has extensive experience in the generation of custom genetically humanized mouse models. With the development of over 200 genetically humanized mouse models and access to the broadest portfolio of genetic engineering technologies, Taconic scientists have unparalleled knowledge about which methods have the greatest chance for success in achieving faithful expression of a particular human gene in a mouse setting. Creating a humanized mouse is not a trivial process and Taconic prides itself with strict adherence to quality control during the entire process of model generation. From thorough analysis of the targeted loci and careful vector design to the molecular analysis of human gene integration, Taconic ensures that clients will obtain reliable data from the humanized model. To further aid in obtaining meaningful data, Taconic can provide services for the expression analysis of the humanized allele and rapid cohort expansion to generate large numbers of experimental animals for your research. A standard humanization project at Taconic takes approximately 40-42 weeks to complete from vector construction, creation and validation of targeted ES cells and production of chimeras. In some cases this timeline may be extended depending on the complexity of the humanization to be achieved. Potential clients interested in the generation of genetically humanized mouse models can inquire with our Custom Model Generation Solutions team who will help assess the feasibility of the project. OUTLOOK Significant advances in genetic engineering technologies over the past 3 decades has led to the generation of genetically engineered humanized mouse models that serve as important tools for a variety of research applications. As technology continues to progress, the degree to which the mouse genome can be humanized will be further realized. Emerging technologies such as CRISPR gene editing have shown great potential for the rapid generation of genetically engineered mice51,52. Although genetic humanization using the CRISPR system has yet to be demonstrated, and size limitations may influence the extent of humanization that can be achieved, gene editing will undoubtedly play an important role in the generation of humanized mouse models. Gene editing may be particularly useful for introducing human genes or variants into 10 TACONIC BIOSCIENCES existing humanized mouse lines. In addition, the efficiency of gene editing may be exploited to introduce knockout mutations on existing humanized mouse lines that further improve their utility and avoids the time consuming process of breeding onto a null background. For instance, introduction of the FRG mutations, which induces a lethal but controllable liver toxicity, into mice harboring humanized genes of the drug metabolism and disposition pathways would allow for humanization of the liver at the cellular and genetic level. Given the ethical and technical constraints associated with human study, model organisms such as genetically humanized mice hold great value and promise in biomedical research. Through a better understanding of human gene function in an in vivo setting, these models are poised to play a key role in the identification and development of novel therapies for the treatment of human disease. 11 TACONIC BIOSCIENCES REFERENCES 1. Devoy, A., Bunton-Stasyshyn, R. K. A., Tybulewicz, V. L. J., Smith, A. J. H. & Fisher, E. M. C. Genomically humanized mice: technologies and promises. Nat. Rev. Genet. 13, 14–20 (2012). 2. Stahl, P. D. & Wainszelbaum, M. J. Human-specific genes may offer a unique window into human cell signaling. Sci. 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One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–8 (2013). 52. Yang, H. et al. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154, 1370–9 (2013). 15 TACONIC BIOSCIENCES Taconic forward thinking. To get you further with your study. ADDITIONAL SERVICES CUSTOM MODEL GENERATION HUMANIZATION CUSTOM BREEDING Taconic Custom Model Generation Solutions empower our clients to develop research models specifically suited to the unique needs of their discovery and development studies or therapeutic programs. Taconic offers both genetic and cell and tissue based humanization of mouse models. Humanized mouse models are increasingly being utilized for a variety of research applications. 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