Transgenic Marmosets Research Articles

Optimization of piggyBac transposon-mediated gene transfer method in common marmoset embryos.

Generating non-human primate models of human diseases is important for the development of therapeutic strategies especially for neurodegenerative diseases. The common marmoset has attracted attention as a new experimental animal model, and many transgenic marmosets have been produced using lentiviral vector-mediated transgenesis. However, lentiviral vectors have a size limitation of up to 8 kb in length for transgene applications. Therefore, the present study aimed to optimize a piggyBac transposon-mediated gene transfer method in which transgenes longer than 8 kb were injected into the perivitelline space of marmoset embryos, followed by electroporation. We constructed a long piggyBac vector carrying the gene responsible for Alzheimer's disease. The optimal weight ratio of the piggyBac transgene vector to the piggyBac transposase mRNA was examined using mouse embryos. Transgene integration into the genome was confirmed in 70.7% of embryonic stem cells established from embryos injected with 1000 ng of transgene and transposase mRNA. Under these conditions, long transgenes were introduced into marmoset embryos. All embryos survived after transgene introduction treatment, and transgenes were detected in 70% of marmoset embryos. The transposon-mediated gene transfer method developed in this study can be applied to the genetic modification of non-human primates, as well as large animals.

Multimodal analyses of a non-human primate model harboring mutant amyloid precursor protein transgenes driven by the human EF1α promoter.

Alzheimer’s disease (AD) is the leading cause of dementia which afflicts tens of millions of people worldwide. Despite many scientific progresses to dissect the AD’s molecular basis from studies on various mouse models, it has been suffered from evolutionary species differences. Here, we report generation of a non-human primate (NHP), common marmoset model ubiquitously expressing Amyloid-beta precursor protein (APP) transgenes with the Swedish (KM670/671NL) and Indiana (V717F) mutations. The transgene integration of generated two transgenic marmosets (TG1&TG2) was thoroughly investigated by genomic PCR, whole-genome sequencing, and fluorescence in situ hybridization. By reprogramming, we confirmed the validity of transgene expression in induced neurons in vitro. Moreover, we discovered structural changes in specific brain regions of transgenic marmosets by magnetic resonance imaging analysis, including in the entorhinal cortex and hippocampus. In immunohistochemistry, we detected increased Aβ plaque-like structures in TG1 brain at 7 years old, although evident neuronal loss or glial inflammation was not observed. Thus, this study summarizes our attempt to establish an NHP AD model. Although the transgenesis approach alone seemed not sufficient to fully recapitulate AD in NHPs, it may be beneficial for drug development and further disease modeling by combination with other genetically engineered models and disease-inducing approaches.

The Common Marmoset-Biomedical Research Animal Model Applications and Common Spontaneous Diseases.

Marmosets are becoming more utilized in biomedical research due to multiple advantages including (1) a nonhuman primate of a smaller size with less cost for housing, (2) physiologic similarities to humans, (3) translatable hepatic metabolism, (4) higher numbers of litters per year, (5) genome is sequenced, molecular reagents are available, (6) immunologically similar to humans, (7) transgenic marmosets with germline transmission have been produced, and (8) are naturally occurring hematopoietic chimeras. With more use of marmosets, disease surveillance over a wide range of ages of marmosets has been performed. This has led to a better understanding of the disease management of spontaneous diseases that can occur in colonies. Knowledge of clinical signs and histologic lesions can assist in maximizing the colony's health, allowing for improved outcomes in translational studies within biomedical research. Here, we describe some basic husbandry, biology, common spontaneous diseases, and animal model applications for the common marmoset in biomedical research.

If I do A, B will happen: Dissecting circuits detecting causal relations between actions and outcomes in marmoset prefrontal cortex

In this issue of Neuron, Duan etal. (2021) use pharmacological manipulation to reveal opposing influences of anterior cingulate and orbitofrontal cortex of marmosets on decisions that are based on action-outcome associations.

Motility Profile of Captive-Bred Marmosets Revealed by a Long-Term In-Cage Monitoring System.

A quantitative evaluation of motility is crucial for studies employing experimental animals. Here, we describe the development of an in-cage motility monitoring method for new world monkeys using off-the-shelf components, and demonstrate its capability for long-term operation (e.g., a year). Based on this novel system, we characterized the motility of the common marmoset over different time scales (seconds, hours, days, and weeks). Monitoring of seven young animals belonging to two different age groups (sub-adult and young-adult) over a 231-day period revealed: (1) strictly diurnal activity (97.3% of movement during daytime), (2) short-cycle (∼20 s) transition in activity, and (3) bimodal diurnal activity including a “siesta” break. Additionally, while the mean duration of short-cycle activity, net daily activity, and diurnal activity changed over the course of development, 24-h periodicity remained constant. Finally, the method allowed for detection of progressive motility deterioration in a transgenic marmoset. Motility measurement offers a convenient way to characterize developmental and pathological changes in animals, as well as an economical and labor-free means for long-term evaluation in a wide range of basic and translational studies.

Generation and Breeding of EGFP-Transgenic Marmoset Monkeys: Cell Chimerism and Implications for Disease Modeling.

Genetic modification of non-human primates (NHP) paves the way for realistic disease models. The common marmoset is a NHP species increasingly used in biomedical research. Despite the invention of RNA-guided nucleases, one strategy for protein overexpression in NHP is still lentiviral transduction. We generated three male and one female enhanced green fluorescent protein (EGFP)-transgenic founder marmosets via lentiviral transduction of natural preimplantation embryos. All founders accomplished germline transmission of the transgene by natural mating, yielding 20 transgenic offspring together (in total, 45 pups; 44% transgenic). This demonstrates that the transgenic gametes are capable of natural fertilization even when in competition with wildtype gametes. Importantly, 90% of the transgenic offspring showed transgene silencing, which is in sharp contrast to rodents, where the identical transgene facilitated robust EGFP expression. Furthermore, we consistently discovered somatic, but so far, no germ cell chimerism in mixed wildtype/transgenic litters. Somatic cell chimerism resulted in false-positive genotyping of the respective wildtype littermates. For the discrimination of transgenic from transgene-chimeric animals by polymerase chain reaction on skin samples, a chimeric cell depletion protocol was established. In summary, it is possible to establish a cohort of genetically modified marmosets by natural mating, but specific requirements including careful promoter selection are essential.

The polymicrogyria-associated GPR56 promoter preferentially drives gene expression in developing GABAergic neurons in common marmosets

GPR56, a member of the adhesion G protein-coupled receptor family, is abundantly expressed in cells of the developing cerebral cortex, including neural progenitor cells and developing neurons. The human GPR56 gene has multiple presumptive promoters that drive the expression of the GPR56 protein in distinct patterns. Similar to coding mutations of the human GPR56 gene that may cause GPR56 dysfunction, a 15-bp homozygous deletion in the cis-regulatory element upstream of the noncoding exon 1 of GPR56 (e1m) leads to the cerebral cortex malformation and epilepsy. To clarify the expression profile of the e1m promoter-driven GPR56 in primate brain, we generated a transgenic marmoset line in which EGFP is expressed under the control of the human minimal e1m promoter. In contrast to the endogenous GPR56 protein, which is highly enriched in the ventricular zone of the cerebral cortex, EGFP is mostly expressed in developing neurons in the transgenic fetal brain. Furthermore, EGFP is predominantly expressed in GABAergic neurons, whereas the total GPR56 protein is evenly expressed in both GABAergic and glutamatergic neurons, suggesting the GABAergic neuron-preferential activity of the minimal e1m promoter. These results indicate a possible pathogenic role for GABAergic neuron in the cerebral cortex of patients with GPR56 mutations.

Generation of Common Marmoset Model Lines of Spinocerebellar Ataxia Type 3

Animal models are indispensable tools in the development of innovative treatments for rare and incurable diseases. To date, there is almost no effective treatment for neurodegenerative diseases, and animal models that properly simulate human disease pathologies are eagerly anticipated to identify disease biomarkers and develop therapeutic methods and agents. Among experimental animals, non-human primates are the most suitable animal models for the study of neurodegenerative diseases with human-specific higher brain dysfunction and late-onset and slowly progressing symptoms. With the rapid development of novel therapies such as oligonucleotide therapeutics and genome editing technologies, non-human primate models for neurodegenerative diseases will be essential for preclinical studies and active interventional trials. In a previous publication, we reported the generation of the first transgenic marmoset model of spinocerebellar ataxia type 3 and successful obtainment of subsequent generations with stable disease onset. Moreover, we generated transgenic marmosets in which the transgene was controlled by the tetracycline-inducible gene expression system. In this mini-review, we summarize the research on our marmoset model of spinocerebellar ataxia type 3.

Digital gene atlas of neonate common marmoset brain

Interest in the common marmoset (Callithrix jacchus) as a primate model animal has grown recently, in part due to the successful demonstration of transgenic marmosets. However, there is some debate as to the suitability of marmosets, compared to more widely used animal models, such as the macaque monkey and mouse. Especially, the usage of marmoset for animal models of human cognition and mental disorders, is still yet to be fully explored. To examine the prospects of the marmoset model for neuroscience research, the Marmoset Gene Atlas (https://gene-atlas.bminds.brain.riken.jp/) provides a whole brain gene expression atlas in the common marmoset. We employ in situ hybridization (ISH) to systematically analyze gene expression in neonate marmoset brains, which allows us to compare expression with other model animals such as mouse. We anticipate that these data will provide sufficient information to develop tools that enable us to reveal marmoset brain structure, function, cellular and molecular organization for primate brain research.

Generation of transgenic marmosets using a tetracyclin-inducible transgene expression system as a neurodegenerative disease model.

Controllable transgene expression systems are indispensable tools for the production of animal models of disease to investigate protein functions at defined periods. However, in nonhuman primates that share genetic, physiological, and morphological similarities with humans, genetic modification techniques have not been well established; therefore, the establishment of novel transgenic models with controllable transgene expression systems will be valuable tools to understand pathological mechanism of human disease. In the present study, we successfully generated transgenic marmosets using a tetracyclin-inducible transgene expression (tet-on) system as a neurodegenerative disease model. The mutant human ataxin 3 gene controlled by the tet-on system was introduced into marmoset embryos via lentiviral transduction, and 34 transgene-introduced embryos were transferred into the uteri of surrogate mothers. Seven live offspring (TET1-7) were obtained, of which four were transgenic. Fibroblasts from TET1 and 3 revealed that inducible transgene expression had occurred after treatment with 10 μg/mL of doxycycline, while treatment with doxycycline via drinking water resulted in 1.7- to 1.8-fold inducible transgene expression compared with before treatment. One transgenic second-generation offspring (TET3-3) was obtained from TET3, and doxycycline-inducible transgene expression in its fibroblasts showed that TET3-3 maintained a high transgene expression level that matched its parent. In conclusion, we established a novel transgenic marmoset line carrying the mutant human ataxin 3 gene controlled by the tet-on system. The development of nonhuman primate models with controllable transgene expression systems will be useful for the identification of disease biomarkers and evaluation of the efficacy and metabolic profiles of therapeutic candidates.

Practical Reproductive Techniques for the Common Marmoset

Abstract: The common marmoset (Callithrix jacchus) is commonly used as a subject model animal in experimental research. The species has several advantages compared with other laboratory primates and we succeeded in creating a transgenic (Tg) marmoset with germline transmission of the transgene, the first time in a nonhuman primate. We have been attempting to further improve marmoset reproductive technology, which is more similar to that of humans than rodent experimental animals, such as mice. We have produced many genetically modified marmosets as human disease models and have also improved marmoset reproductive techniques to obtain many fertilized embryos and neonates. For ethical reasons, it is difficult to perform human reproductive studies; thus, we must rely on nonhuman primate models in basic research. For this reason, reproductive studies of marmosets may help the development of assisted reproduction technologies (ART) for humans and may also be useful in human preclinical studies. In this minisym...

Transgenic Monkey Model of the Polyglutamine Diseases Recapitulating Progressive Neurological Symptoms.

Age-associated neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, and the polyglutamine (polyQ) diseases, are becoming prevalent as a consequence of elongation of the human lifespan. Although various rodent models have been developed to study and overcome these diseases, they have limitations in their translational research utility owing to differences from humans in brain structure and function and in drug metabolism. Here, we generated a transgenic marmoset model of the polyQ diseases, showing progressive neurological symptoms including motor impairment. Seven transgenic marmosets were produced by lentiviral introduction of the human ataxin 3 gene with 120 CAG repeats encoding an expanded polyQ stretch. Although all offspring showed no neurological symptoms at birth, three marmosets with higher transgene expression developed neurological symptoms of varying degrees at 3–4 months after birth, followed by gradual decreases in body weight gain, spontaneous activity, and grip strength, indicating time-dependent disease progression. Pathological examinations revealed neurodegeneration and intranuclear polyQ protein inclusions accompanied by gliosis, which recapitulate the neuropathological features of polyQ disease patients. Consistent with neuronal loss in the cerebellum, brain MRI analyses in one living symptomatic marmoset detected enlargement of the fourth ventricle, which suggests cerebellar atrophy. Notably, successful germline transgene transmission was confirmed in the second-generation offspring derived from the symptomatic transgenic marmoset gamete. Because the accumulation of abnormal proteins is a shared pathomechanism among various neurodegenerative diseases, we suggest that this new marmoset model will contribute toward elucidating the pathomechanisms of and developing clinically applicable therapies for neurodegenerative diseases.

The marmoset as a model system for studying voluntary motor control.

The common marmoset has recently gained interest as an animal model for systems and behavioral neuroscience. This is due in part to the advent of transgenic marmosets, which affords the possibility of combining genetic manipulations with physiological recording and behavioral monitoring to study neural systems. In this review, they will argue that the marmoset provides a unique opportunity to study the neural basis of voluntary motor control from an integrative perspective. First, as an intermediate animal model, the marmoset represents an important bridge in motor system function between other primates, including humans, and rodents. Second, due to the marmoset's small brain size and lissencephalic cortex, novel electrophysiological and optical recording technologies will allow an integrative study of cortical function at multiple spatial scales beyond that afforded by other non-human primate models. Finally, as a primate expressing an ancestral state of corticospinal organization, the marmoset offers the possibility of understanding the integrative function of cortical and spinal interneuron circuitry in isolation of more recent corticomotoneuronal elaborations. If the potential of the marmoset as a model species is to be realized, they will need to learn to work with their natural behavioral repertoire. They have concluded by considering practical aspects of studying motor systems with marmosets. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 273-285, 2017.

Generation of transgenic marmosets expressing genetically encoded calcium indicators.

Chronic monitoring of neuronal activity in the living brain with optical imaging techniques became feasible owing to the continued development of genetically encoded calcium indicators (GECIs). Here we report for the first time the successful generation of transgenic marmosets (Callithrix jacchus), an important nonhuman primate model in neurophysiological research, which were engineered to express the green fluorescent protein (GFP)-based family of GECIs, GCaMP, under control of either the CMV or the hSyn promoter. High titer lentiviral vectors were produced, and injected into embryos collected from donor females. The infected embryos were then transferred to recipient females. Eight transgenic animals were born and shown to have stable and functional GCaMP expression in several different tissues. Germline transmission of the transgene was confirmed in embryos generated from two of the founder transgenic marmosets that reached sexual maturity. These embryos were implanted into six recipient females, three of which became pregnant and are in advanced stages of gestation. We believe these transgenic marmosets will be invaluable non-human primate models in neuroscience, allowing chronic in vivo monitoring of neural activity with functional confocal and multi-photon optical microscopy imaging of intracellular calcium dynamics.

357. Blood-Brain Interface Opening By Ultrasound in Adult Common Marmoset To Induce Brain Pathology With rAAV

Background: The common marmoset (Callithrix jacchus) could provide an appropriate model for neuromuscular diseases because of its higher brain function and physiological resemblance to human. Although production of transgenic marmosets from zygote transduced with lentiviral vector were reported, this strategy is too costly and not efficient with various reasons. In contrast, an adeno-associated virus vector (rAAV) has great advantages to induce pathology in the aged animals. However, fully maturated blood-brain interface (BBI) significantly limits passive rAAV transport from circulation to the brain. To overcome this issue, we investigated BBI opening, which is a promising technology to make cerebral capillary open by transiently disrupting tight junction in BBI with ultrasound irradiation (UI) just after i.v. injection of microbubble (MB).Methods: Commercially available MB or homemade bubbleliposome (BL) mixed with evans blue were i.v. injected into the femoral vein of the anesthetized adult marmosets. rAAV1 was also injected simultanously in certain trials. Subsequently, transcranial (TC) UI to the brain was performed for several minutes. The brain was removed 24 hrs after the UI. To visualize the leakage of albumin (ALB) in more detail, cryosections were immunologically stained with antibodies against ALB and CD31. Thin coronal slice of the brain was pieced into various parts, and genome DNA was extracted from the each pieces. Then relative rAAV genome copies were measured by qPCR. For live imaging analysis, BL mixed with carbon 11-labeled aminoisobutyric acid were i.v. injected into the tail vein. Subsequently, TCUI and positron emission tomography (PET) scan were performed.Results: In TCUI from the temporal region, ALB mainly leaked at the temporal cortex and hippocampus. In TCUI from circumference, ALB leakage seemed to occur at whole brain except for white matter.Microscopically, ALB leakage was found around the blood vessels of 10-50 mm in diameter. Especially, leakage in hippocampus was prominent. Relative rAAV genome copies were imbalanced between symmetrical piece pair. PET revealed ALB leakage not only in hippocampus but also in the anterior cingulate cortex, basal ganglia, optic tract, amygdala, and superior temporal gyrus.Conclusion: UI-mediated MB-assisted TC-BBI opening is promising approach in the generation of the disease model based on adult common marmoset.

Reprogramming non-human primate somatic cells into functional neuronal cells by defined factors

BackgroundThe common marmoset (Callithrix jacchus) is a New World primate sharing many similarities with humans. Recently developed technology for generating transgenic marmosets has opened new avenues for faithful recapitulation of human diseases, which could not be achieved in rodent models. However, the longer lifespan of common marmosets compared with rodents may result in an extended period for in vivo analysis of common marmoset disease models. Therefore, establishing rapid and efficient techniques for obtaining neuronal cells from transgenic individuals that enable in vitro analysis of molecular mechanisms underlying diseases are required. Recently, several groups have reported on methods, termed direct reprogramming, to generate neuronal cells by defined factors from somatic cells of various kinds of species, including mouse and human. The aim of the present study was to determine whether direct reprogramming technology was applicable to common marmosets.ResultsCommon marmoset induced neuronal (cjiN) cells with neuronal morphology were generated from common marmoset embryonic skin fibroblasts (cjF) by overexpressing the neuronal transcription factors: ASCL1, BRN2, MYT1L and NEUROD1. Reverse transcription-polymerase chain reaction of cjiN cells showed upregulation of neuronal genes highly related to neuronal differentiation and function. The presence of neuronal marker proteins was also confirmed by immunocytochemistry. Electrical field stimulation to cjiN cells increased the intracellular calcium level, which was reversibly blocked by the voltage-gated sodium channel blocker, tetrodotoxin, indicating that these cells were functional. The neuronal function of these cells was further confirmed by electrophysiological analyses showing that action potentials could be elicited by membrane depolarization in current-clamp mode while both fast-activating and inactivating sodium currents and outward currents were observed in voltage-clamp mode. The 5-bromodeoxyuridine (BrdU) incorporation assay showed that cjiN cells were directly converted from cjFs without passing a proliferative state.ConclusionsFunctional common marmoset neuronal cells can be obtained directly from embryonic fibroblasts by overexpressing four neuronal transcription factors under in vitro conditions. Overall, direct conversion technology on marmoset somatic cells provides the opportunity to analyze and screen phenotypes of genetically-modified common marmosets.

Top row from the left: Knock in in Silkworm, see Daimon et al., 14–25. Knock out in Xenopus laevis, see Suzuki et al., 108–114. Knock out in zebrafish, see Kawahara et al., 26–33. Knock out in medaka, see Kinoshita et al., 98–107. Middle row: Schematic drawings of the principal mechanisms of Genome editing by Zinc‐finger nucleases method, TAL effector nucleases

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Common marmoset as a new model animal for neuroscience research and genome editing technology

The common marmoset (Callithrix jacchus) is a small New World primate; it originally comes from the Atlantic coastal forests in northeastern Brazil. It has been attracting much attention in the biomedical research field because of its size, availability, and unique biological characteristics. Its endocrinological and behavioral similarity to humans, comparative ease in handling, and high reproductive efficiency are very advantageous for neuroscience research. Recently, we developed transgenic common marmosets with germline transmission, and this technological breakthrough provides a potential paradigm shift by enabling researchers to investigate complex biological phenomena using genetically-modified non-human primates. In this review, we summarize recent progress in marmoset research, and also discuss a potential application of genome editing tools that should be useful toward the generation of knock-out/knock-in marmoset models.

Effect of the size of zona pellucida opening on hatching in the common marmoset monkey (Callithrix jacchus) embryo

The use of the common marmoset monkey in biomedical research has increased recently, and further attention has been devoted to this model after the successful production of transgenic marmosets. To extend genetic engineering approaches to widespread biomedical research fields, efficient prolonged in vitro culturing of embryo development is necessary. We aimed to evaluate the effects of the size of the zona pellucida opening on promoting the hatching process in the marmoset embryo. Piezo-microdrilling of a 6-μm opening in eight embryos resulted in four partially hatched embryos and one hatched embryo after 5 days of culture. Piezo-microdrilling a 20-μm opening in 11 embryos resulted in nine partial hatchings and no hatched embryos. Piezo-scraping an 80-μm opening in six embryos resulted in no partially hatched embryos and five hatched embryos. These results suggest that an 80-μm opening, rather than 6-μm or 20-μm openings, is suitable to complete the hatching process in the marmoset embryo.

Transgenic Animals, Biomedical Experiments, and "Progress"

Abstract By conducting a critical discourse analysis of a scientific research article that claims additional potential for using transgenic marmosets in biomedical experiments, this article critiques instrumental approaches to scientific progress as they are expressed in scientific research that uses nonhuman animal experiments. Following an analysis that focuses on issues associated with access to publication, assertions about scientific breakthrough and scientific facts, and the construction of science as impartial, the article concludes that manipulating the genetics of nonhuman animals to engineer a predisposition to the development of feared human health hazards represents moral deterioration rather than progress.