The genetically altered ES cells can populate all the tissues of the developing mouse from the blastocyst stage. The contribution of the ES cells to the genetic makeup of the chimeric animal that develops from the injected blastocyst is most easily assessed by using ES cells and blastocysts whose genes for coat color differ. If the ES cells contribute to the germ cells of the developing mouse embryo, their entire haploid genome can be passed to subsequent generations. The isolation of ES cell line and the demonstration that transfected DNA can recombine with its homologous chromosome counterpart in the genome in the mammalian cell lines, including ES cells, are the 2 most important observations that led to the production of gene knockout animals.
The ES cells are derived from the inner cell mass of a blastocyst and can be kept indefinitely in culture without jeopardizing their totipotency and can be reintroduced into a host embryo by either injection into blastocyst cavity or aggregation of ES cells with morulae.
Both methods are successfully used to produce chimeric animals in which manipulated ES cells have contributed to somatic tissue and germ line lineage. A common approach to disrupt gene function by homologous recombination in ES cells is to construct a vector targeting vector or knockout vector designed to undergo a homologous recombination with its chromosomal counterpart.
The goal of the gene-targeting knockout method is to replace a specific gene of interest with another one that is inactive, altered, or irrelevant. Since the integration of the knockout vector into a random chromosomal site is much more frequent than into its homologous site in the genome, several techniques are designed to select cells with site-specific recombinant events.
To increase the probability of site-specific replacement, both ends of the replacement gene are flanked by long DNA sequences that are homologous to the sequences that flank the target gene. Gene constructs of this type permit corresponding stretches of the DNA to be exchanged this is termed homologous recombination when the DNA breaks and rejoins. The frequency of the homologous recombination is very low. Therefore, there must be a way to select the rare cells in which the target gene has been replaced by the constructed gene. The 2 strategies to select specific cells, positive and negative selection, are illustrated in Figure 2.
After the targeting construct is delivered into the ES cells, the recombinant cells can be selected based on the targeting construct design. To date, all targeted mutations introduced into the germ line have been created using a selectable marker, usually neomycin resistance and occasionally hygromycin resistance, to disrupt the coding sequence of the targeted gene. To enrich for targeted events, the most widely used strategy is the positive or negative selection approach that can enrich 2- to fold.
In the construct, the bacterial neomycin resistance neor gene disrupts the coding sequence of the mouse gene. In addition, the herpes simplex virus thymidine kinase HSV-tK gene is placed at one or both ends of the targeting construct. The antibiotic G is used to select for cells in which the DNA construct containing the neor gene has been integrated, either randomly or by homologous recombination.
Thus, cells that contain random integration into a chromosomal site that allow expression of the HSV-tK gene will be killed. By positive or negative selection strategy, the cells with targeted integration with the neor gene but without the HSV-tK gene will be selected. The selected ES cells cells are injected into the blastocele cavity of the blastocysts at around 3.
The injected blastocysts are transferred to uteri of the pseudopregnant recipient female mice. Generally, 4 to 16 blastocysts are transferred to the uterine horns. The pups are usually born 17 days after the transfer.
Through a crossbreeding and selection process, heterozygous and homozygous knockout mice can be generated. Many neurodegenerative disorders or subsets of these disorders, such as amyotrophic lateral sclerosis ALS , Alzheimer disease AD , prion disease, glutamine expansion disorders such as Huntington disease [HD] and spinocerebellar ataxia [SCA] , and spinal muscular atrophy, are inherited as either dominant or recessive traits in humans. The mechanisms by which vulnerable cells dysfunction or die in these disorders are not well understood. There are no known genetic mimics naturally occurring in animals for these diseases.
However, when mutations responsible for the disease in a specific gene are identified in humans, a mouse model can then be generated. Described below are representative mouse models for several neurodegenerative disorders such as ALS, glutamine expansion disorders, AD, and prion disorders. Amyotrophic lateral sclerosis is a fatal neurological disease characterized by the degeneration of the motor neurons in the brain and spinal cord.
In most cases, FALS is inherited as an autosomal dominant trait. In addition, a dominant form of juvenile ALS-like syndrome has been mapped to 9q The copper-zinc superoxide dismutase SOD1 gene on chromosome 21 is the only identified genetic element that when mutated is causative of FALS. SOD1 converts superoxide to form molecular oxygen and hydrogen peroxide, the latter of which is detoxified by catalase and glutathione peroxidase.
Because both superoxide and hydrogen peroxide are toxic to cells, it is speculated that the disturbance of free radical homeostasis, by either an increase or decrease in dismutase activity, may lead to cell death. Initially, when decreased dismutase activity was identified in individuals with mutant SOD1 , it was hypothesized that the decrease rather than increase in dismutase activity may be responsible to the disease.
Although several lines of evidence supported this hypothesis, transgenic mouse overexpressing the mutant SOD1 developed an ALS-like syndrome, which strongly argues for a gain-of-function mechanism. The first mutant transgenic mouse line was developed by overexpression of G93A mutation, a mutation that causes FALS in humans. Wong et al 6 reported that transgenic mice that overexpressed G37R mutation of SOD1 developed severe, progressive motor neuron disease. The most obvious cellular abnormality in G93A mice and the G37R mice is the presence of membrane-bound vacuoles in axons, dendrites, and cell bodies.
This pathological aspect is not a common feature of human disease, but ALS mice expressing the lower amount of mutant SOD1 have an abnormality akin to that seen in humans. The G86R mice also showed degenerative changes of motor neurons within the spinal cord, brainstem, and neocortex. Common to all 3 lines of mutant SOD1 transgenic mice is that the mice with the higher copy number of the mutant SOD1 gene and with higher SOD1 activity become paralyzed earlier, providing strong evidence that onset of disease is influenced by dose of mutant SOD1 protein and that decreased SOD1 activity is not the primary care of ALS.
Further support of this observation comes from SOD1 knockout mice. Reaume et al 8 generated SOD1 knockout mice using homologous recombination. They found that mice lacking SOD1 developed normally and that 1-year-old mice show no overt motor deficit.
These mice, however, do have increased motor neuron loss after axonal injury and distal axonal loss with age. These results indicate that SOD1 is not necessary for normal motor neuron development and function but may be required in physiologically stressful conditions or those following injury. Numerous hypotheses have been proposed and some tested in ALS mice. By crossbreeding mice that overexpress bcl-2 to SOD1-G93A transgenic mice, Kostic et al 9 found that overexpression of the antiapoptotic protein bcl-2 delayed onset of ALS and prolonged survival in G93A mice, although the duration of the disease was not altered.
This finding was further expanded by Pasinelli et al. Proteolytic processing characteristic of caspase-1 activation is seen both in spinal cords of transgenic ALS mice and neurally differentiated neuroblastoma cells with SOD1 mutations.
Clinical Genetics 57, Rosenthal, N. Animal models of depression In accordance with the first theory of depression, the so-called monoaminergic hypothesis, many studies have demonstrated that patients with major depression have abnormalities in the neurotransmitters of the brain, particularly serotonin 5-hydroxytryptamine, 5-HT , noradrenaline, and, as demonstrated more recently, dopamine DA. From bedside to bench and back again: research issues in animal models of human disease. Select Parent Grandparent Teacher Kid at heart. See S3 Table for associated statistical analyses in supporting information. Mouse models for chromosome 9 hexanucleotide repeat 72 C9ORF72 -related amyotrophic lateral sclerosis. J Neurosci 29 41 : - ,
Cytoplasmic Lewy body—like hyaline inclusions are found in motor neurons of patients with ALS. The SOD1-positive inclusions were also identified in transgenic mice expressing SOD1-G93A, raising the possibility that the formation of these inclusions may interfere in the neuronal cell function and lead to motor neuron death in FALS. They found that the presence of SOD1-positive aggregates was a common finding of the disease regardless of the presence or absence of endogenous SOD1. These mice develop the typical phenotype and pathologic features of ALS.
SOD1 inclusions can be detected in neurons and neuritic processes in the gray matter of the spinal cord and brainstem, suggesting that an altered form of mutant SOD1 may participate in the pathogenesis of FALS. This mouse probably provides the minimal lesion necessary for neurodegeneration and provides evidence that a truncated SOD1 polypeptide is sufficient to cause disease.
Neurofilaments are another suspected component involved in the pathogenesis of ALS. It was reported that overexpression of neurofilament subunits NF-L or NF-H in transgenic mice produced morphologic alterations that resemble the pathological features of human ALS, although without extensive motor neuron death.
To investigate how neurofilaments might cause neuropathy and its relevance to ALS, several groups used transgenic mouse models and reported some interesting results. Taken together, these results indicate that although disorganized neurofilaments can sometimes cause neuropathy, neurofilaments are not required for mutant SOD1-mediated neurodegeneration; rather absence of neurofilaments from the axons may play a protective role in this process.
Several findings make this group of diseases unique. First, these disorders are characterized by "anticipation. Second, the age of onset may be inversely correlated with the number of CAG repeats. Third, these genes are not homologous except that they all share CAG expansion. Fourth, although the expression of the genes is widespread, each disorder has highly selective but overlapping degeneration of different neurons, such as striatal neurons in HD, cerebellar Purkinje cells in SCAs, and brainstem and spinal motor neurons in spinobulbar muscular atrophy. The mechanisms by which the CAG expansions cause these glutamine expansion disorders are not known.
In the past several years, transgenic studies have provided substantial information in this field. Huntington disease is a devastating neurological disorder associated with progressive chorea, rigidity, and dementia. It usually manifests in midlife and results in selective neuron loss that is most prominent in the striatum and basal ganglia.
The HD gene encodes a ubiquitously expressed protein huntingtin containing a glutamine repeat. The number of repeats varies from 8 to 35 in healthy individuals. Individuals develop HD if the repeat number is more than To understand the normal function of the HD gene, 3 groups have independently created knockout mice using homologous recombination.
They found that homozygous knockout mice showed developmental retardation and died around 8 days. The heterozygous mice displayed increased motor activity and cognitive deficits.
Because neither homozygous nor heterozygous HD knockout mice recapitulate the phenotype and neuropathologic features of HD, it follows that the HD gene, although essential for postimplantation development and normal functioning of the basal ganglia, does not cause HD due to loss of the intrinsic function.
A gain-of-function mechanism is therefore postulated for the pathogenesis of HD. To investigate the role of glutamine expansion of huntingtin in HD, Mangiarini et al 18 established a transgenic mouse model using a construct that contains a 1. The transgenic mice exhibited a progressive neurological phenotype that resembles many of the features of HD, including choreiform-like movements, involuntary stereotypic movements, tremor, and epileptic seizures. Because this transgene only expressed a small, glutamine-containing portion of huntingtin and still led to the disease, it was suggested that expression of a truncated amino-terminal fragment of huntingtin with a greatly expanded glutamine repeat is sufficient to cause a progressive neurological phenotype resembling HD.
SCA1 is characterized by progressive ataxia and selective neuronal loss within the cerebellar cortex and brainstem. The normal function of the SCA1 gene is not known. To gain insight into the function of this gene, Matilla et al 19 generated SCA1 knockout mice by deletion of exon 8, which contains most of the coding region of the SCA1 gene.
The mice lacking ataxin-1 did not show any evidence of ataxia or neurodegeneration, although the results suggested that ataxin-1 plays a role in learning and memory. To understand the pathogenesis of SCA1, Burright et al 20 established a transgenic mouse model by overexpression of human SCA1-coding region containing 82 CAG repeats using the regulatory region of the Purkinje cell—specific gene Pcp2.
The transgenic mice with the expanded SCA1 allele developed ataxia and Purkinje cell degeneration, indicating that expanded CAG repeats expressed in Purkinje cells are sufficient to produce ataxia. This observation has led to the widely held view that these abnormal depositions are toxic and may be pathogenic. The first series overexpressed ataxin-1 containing 82 CAG repeats ataxin-1  with a mutated nuclear localization signal KT. The ataxin-1 82 with KT is exclusively distributed in cytoplasm but not in the nucleus.
They found that without nuclear distribution of ataxin-1 82 , the mice did not exhibit ataxia, NIIs, or pathological changes. This model demonstrated that nuclear localization of the ataxin-1 is critical for pathogenesis. The second series overexpressed ataxin-1 77 containing a deletion within the self-association region in Purkinje cell nuclei. These mice developed ataxia and Purkinje cell abnormalities similar to the original SCA1 mice. However, no evidence of NIIs was found. Thus, they concluded that although nuclear localization of ataxin-1 is necessary for the development of the disease, NIIs of ataxin-1 are not required to initiate the pathogenesis in the transgenic mice.
Similar results were also obtained by Saudou et al. Blocking nuclear localization of the mutant huntingtin suppressed its ability to form NIIs and to induce neurodegeneration. However, the formation of NIIs did not correlate with huntingtin-induced cell death. Conversely, suppression of the NIIs formation resulted in an increase in mutant huntingtin—induced cell death, suggesting NII formation may reflect a cellular mechanism to protect against huntingtin-induced cell death. Recently, Cummings et al 23 presentedevidence that although the pathogenic role of small, submicroscopic aggregates still remains possible, the large inclusions are not required for expanded polyglutamine pathology.
Alzheimer disease is the fourth leading cause of death in the United States. The presence of the apolipoprotein E4 apoE4 allele is a robust risk factor for late-onset AD. After the identification of the pathogenic mutations in the APP gene, many investigators tried to develop a mouse model. Partly due to the technical difficulties in handling the large APP gene approximately kilobases , transgenes were made with APP cDNA sequences and a variety of promoters. Unfortunately, these transgenic mice failed to show extensive type neuropathology and phenotype.
In , Games et al 24 constructed a hybrid transgene, which includes the full-length human APP cDNA with ValPhe mutation under the control of the platelet-derived growth factor promoter. Inserted into this transgene are introns 6, 7, and 8 of APP gene, which are important for alternative splicing of the gene product.
Probably due to these changes, this construct drives much higher levels of APP expression than standard cDNA constructs. PS1 and PS2 encode membrane-associated proteins of great similarity; therefore, they may share similar function. Mutations in the PS1 gene account for most early-onset familial AD. The homozygous mutants died shortly after birth and showed multiple defects in skeleton and central nervous system, suggesting that PS1 is essential for proper formation of the axial skeleton, normal neurogenesis, and neuronal survival.
This finding was confirmed and further expanded by in vivo and in vitro experiments. Apolipoprotein E apoE is a kd, glycosylated, lipid-binding protein that mediates the redistribution of lipids among cells. It is highly expressed in brain and liver. Human apoE exists in 3 major isoforms encoded by distinct alleles E2, E3, and E4 on chromosome Clinical evidence suggests that besides age, the apoE4 is the most important known risk factor for the development of late-onset familial and sporadic forms of AD, whereas E2 provides relative protection from this illness.
Although many hypotheses have been proposed, it remains unclear how apoE4 affects cognition and increases the AD risk. To examine the effects of apoE4 on AD, several groups have made apoE knockout mice and transgenic mice expressing different alleles of apoE. By crossbreeding, mice expressing different human apoE isoforms in the absence of mouse endogenous apoE have been established. Although these mice did not show evidence of senile plaques, some mouse lines showed impairment in learning and in vertical exploratory behavior. The prion diseases, sometimes referred to as the transmissible spongiform encephalopathies , include kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker disease of human as well as scrapie and bovine spongiform encephalopathy of animals.
The unique characteristic common to all of these disorders, whether sporadic, dominantly inherited, or acquired by infection, is that they involve the aberrant metabolism of the prion protein PrP. Although much emphasis was placed on the transmissible nature of the diseases for many decades, most solid lines of evidence regarding the pathogenesis are derived from transgenic studies of the inherited form of the diseases.
The discovery of a mutation at codon PL in the PrP gene in a family with Gerstmann-Straussler-Scheinker disease established the genetic basis for prion diseases. To investigate the nature of the diseases, Hsiao et al 31 and Telling et al 32 created a transgenic mouse model that overexpressed a mutant mouse PrP with a substitution P, which corresponds to PL in human mutant PrP identified in Gerstmann-Straussler-Scheinker disease.
The resulting mice spontaneously developed spongiform neurodegeneration between 50 and days of age, while the control lines that overexpressed wild-type PrP remained normal for more than days. Furthermore, they found that when Syrian hamsters or mice were inoculated with brain homogenates from these spontaneously sick transgenic mice, the inoculated animals developed similar disease. However, the control group that was inoculated with brain homogenates from normal mice remained free of symptoms.
These findings indicate that mutant PrP gene not only causes but also produces infectious particles for prion disease. If prion diseases are caused exclusively by prion and not any other agents such as DNA and RNA, the next question would be how prion propagates and what are the substrates for the prion propagation. The "protein only" hypothesis proposes that PrPSc prion protein: scrapie form is the pathogenically modified form of PrPC prion protein: cellular form and PrPSc is devoid of nucleic acid. Incidence is approximately 1 in 10, female births worldwide.
Caused by loss-of-function mutations in a transcription regulator gene that encodes methyl CpG-binding protein 2 MECP2 , currently no effective treatment exists for RTT. In the case of RTT, the therapeutic potential of viral vector—mediated MECP2 gene transfer has been demonstrated in mouse models during preclinical trials. Vectors for Gene Therapy Solutions Several viral vectors have been investigated as possible mechanisms for transgene expression in gene therapy solutions, including adeno-viral vectors, adeno-associated viral AAV vectors, and retroviral vectors such as lentiviral vectors.
Perhaps the most exciting recent development has been the ability to engineer novel AAV capsids with an unprecedented capacity to transfer genes to the CNS of animal models following standard administrations. It is a neurodegenerative disorder with an incidence of 1 in 10, births and is caused by mutations of the survival motor neuron 1 SMN1 gene. Four subtypes of SMA types are recognized, based upon motor milestone achievement and the age at which symptoms become apparent. The phenotype is dependent on the number of copies of a modifier gene, the survival motor neuron 2 SMN2 gene.
A recent open-label study assessed the health outcomes of 12 patients with homozygous deletions of SMN1 and 2 copies of SMN2. Patients were given a single intravenous infusion of an AAV9 vector called Zolgensma AveXis , which contains the human survival motor neuron gene. The outcomes included decreased respiratory and nutritional complications, improved motor milestone achievements, and fewer hospitalizations.
The mouse has many advantages over human beings for the study of genetics, including the unique property that genetic manipulation can be routinely carried. Negative views of animal modeling of neurological diseases often stem from gene by gene studies in mouse models are severely limiting.
Currently, no approved therapies exist to treat AADC deficiency, and best practice is to inhibit monoamine oxidase and to directly stimulate dopamine receptors using dopamine agonists. However, this treatment has little benefit for patients. Motor function was remarkably improved for up to 2 years following treatment. Positron emission tomography using a specific tracer for AADC showed increased uptake of the enzyme within the putamen.