Download The Advantage and Application of Genetically Humanized Mouse

The Advantage
and Application of
Genetically Humanized
Mouse Models for
Biomedical Research
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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
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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
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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
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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
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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
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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.
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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’.
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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.
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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
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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.
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