Population genetics of marmosets in Asian primate research centers and loci associated with epileptic risk revealed by whole-genome sequencing

The common marmoset(Callithrix jacchus)has emerged as a valuable nonhuman primate model in biomedical research with the recent release of high-quality reference genome assemblies.Epileptic marmosets have been independently reported in two Asian primate research centers.Nevertheless,the population genetics within these primate centers and the specific genetic variants associated with epilepsy in marmosets have not yet been elucidated.Here,we characterized the genetic relationships and risk variants for epilepsy in 41 samples from two epileptic marmoset pedigrees using whole-genome sequencing.We identified 14558184 single nucleotide polymorphisms(SNPs)from the 41 samples and found higher chimerism levels in blood samples than in fingernail samples.Genetic analysis showed fourth-degree of relatedness among marmosets at the primate centers.In addition,SNP and copy number variation(CNV)analyses suggested that the WW domain-containing oxidoreductase(WWOX)and Tyrosine-protein phosphatase nonreceptor type 21(PTPN21)genes may be associated with epilepsy in marmosets.Notably,KCTD18-like gene deletion was more common in epileptic marmosets than control marmosets.This study provides valuable population genomic resources for marmosets in two Asian primate centers.Genetic analyses identified a reasonable breeding strategy for genetic diversity maintenance in the two centers,while the case-control study revealed potential risk genes/variants associated with epilepsy in marmosets.

Ancient DNA of the pygmy marmoset type specimen Cebuella pygmaea(Spix,1823)resolves a taxonomic conundrum

The pygmy marmoset,the smallest of the anthropoid primates,has a broad distribution in Western Amazonia.Recent studies using molecular and morphological data have identified two distinct species separated by the Napo and Solimoes-Amazonas rivers.However,reconciling this new biological evidence with current taxonomy,i.e.,two subspecies,Cebuella pygmaea pygmaea(Spix,1823)and Cebuella pygmaea niveiventris(Lönnberg,1940),was problematic given the uncertainty as to whether Spix’s pygmy marmoset(Cebuella pygmaea pygmaea)was collected north or south of the Napo and Solimoes-Amazonas rivers,making it unclear to which of the two newly revealed species the name pygmaea would apply.Here,we present the first molecular data from Spix’s type specimen of Cebuella pygmaea,as well as novel mitochondrial genomes from modern pygmy marmosets sampled near the type locality(Tabatinga)on both sides of the river.With these data,we can confirm the correct names of the two species identified,i.e.,C.pygmaea for animals north of the Napo and Solimoes-Amazonas rivers and C.niveiventris for animals south of these two rivers.Phylogenetic analyses of the novel genetic data placed into the context of cytochrome b gene sequences from across the range of pygmy marmosets further led us to reevaluate the geographical distribution for the two Cebuella species.We dated the split of these two species to 2.54 million years ago.We discuss additional,more recent,subdivisions within each lineage,as well as potential contact zones between the two species in the headwaters of these rivers.

Visual response characteristics of neurons in the second visual area of marmosets

The physiological characteristics of the marmoset second visual area(V2) are poorly understood compared with those of the primary visual area(V1). In this study, we observed the physiological response characteristics of V2 neurons in four healthy adult marmosets using intracortical tungsten microelectrodes. We recorded 110 neurons in area V2, with receptive fields located between 8° and 15° eccentricity. Most(88.2%) of these neurons were orientation selective, with half-bandwidths typically ranging between 10° and 30°. A significant proportion of neurons(28.2%) with direction selectivity had a direction index greater than 0.5. The vast majority of V2 neurons had separable spatial frequency and temporal frequency curves and, according to this criterion, they were not speed selective. The basic functional response characteristics of neurons in area V2 resemble those found in area V1. Our findings show that area V2 together with V1 are important in primate visual processing, especially in locating objects in space and in detecting an object's direction of motion. The methods used in this study were approved by the Monash University Animal Ethics Committee, Australia(MARP 2009-2011) in 2009.

Synchrotron Refraction Enhanced Tomography of an Intact Common Marmoset (<i>Callithrix jacchus</i>)

The common marmoset (Callithrix jacchus), a small new world monkey species, has been widely used in various scientific fields. It is necessary to understand connections between specific genotypes, their structure, and function;however, an anatomical atlas of the entire body of the common marmoset has not yet been reported. In addition to conventional absorption, refraction enhanced computed tomography (CT) based on synchrotron radiation can increase the contrast of boundaries between small absorption differences. In this study, to examine the potential of creating an anatomical atlas of the whole body of the common marmoset non-invasively, we visualized an intact marmoset using synchrotron refraction enhanced CT. The cryogenic marmoset was scanned using the medical imaging beamline at the SPring-8 synchrotron radiation research facility in JAPAN. The trabecular structure, articular cartilage, cruciate ligament in the knee joint, and small airways (diameter: > 400 μm) was clearly identified with 50 μm voxel size and 37 keV x-ray energy. The structure of the heart and branching vessels in the kidneys and liver were also identified without contrast agents, and the anatomical structure of the brain was slightly visible. These results show that synchrotron refraction enhanced CT is useful for creating an anatomical atlas non-invasively, and further studies are planned that will combine refraction enhanced CT and other imaging techniques to analyse the morphology and create a complete atlas of the whole body of the common marmoset.

Developing biomarkers for neurodegenerative diseases using genetically-modified common marmoset models

Mouse and non-human primate models of neurodegenerative disease：The prevalence of age-related neurodegenerative diseases continues to increase with ever increasing aging population over the age of 60.Although the difficulties associated with neurodegenerative diseases present an urgent global issue,there is no effective treatment for these conditions.

Leukemogenic Fusion Gene (p190 BCR-ABL) Transduction into Hematopoietic Stem/Progenitor Cells in the Common Marmoset

Patients with Philadelphia chromosome (p190 BCR-ABL fusion gene)-positive acute lymphoblastic leukemia have a poor prognosis despite intensive therapeutic intervention.In this study, we attempted to develop a leukemia nonhuman primate model that mimics various human systems. Hematopoietic stem/progenitor cells in the common marmoset were transduced with a lentiviral vector containing the p190 BCR-ABL fusion gene by ex vivo transduction or in vivo direct bone marrow injection. In the latter model, BCR-ABL gene expression was maintained for more than one and a half years. One marmoset unexpectedly developed myelofibrosis-like disease. However, none of the marmosets have developed leukemia to date. In conclusion, we successfully achieved sustained p190 BCR-ABL gene expression in vivo. However, a genetic mutation in addition to p190 BCR-ABL may be required for the malignant transformation of hematopoietic stem/progenitor cells in the common marmoset during the short observation period. This novel in vivo approach will help develop a marmoset leukemia model in the future.

Induction of Anxiety-Like Phenotypes by Knockdown of Cannabinoid Type-1 Receptors in the Amygdala of Marmosets

The amygdala is an important hub for regulating emotions and is involved in the pathophysiology of many mental diseases,such as depression and anxiety.Meanwhile,the endocannabinoid system plays a crucial role in regulating emotions and mainly functions through the cannabinoid type-1 receptor(CB1R),which is strongly expressed in the amygdala of non-human primates(NHPs).However,it remains largely unknown how the CB1Rs in the amygdala of NHPs regulate mental diseases.Here,we investigated the role of CB1R by knocking down the cannabinoid receptor 1(CNR1)gene encoding CB1R in the amygdala of adult marmosets through regional delivery of AAV-SaCas9-gRNA.We found that CB1R knockdown in the amygdala induced anxiety-like behaviors,including disrupted night sleep,agitated psychomotor activity in new environments,and reduced social desire.Moreover,marmosets with CB1R-knockdown had up-regulated plasma cortisol levels.These results indicate that the knockdown of CB1Rs in the amygdala induces anxiety-like behaviors in marmosets,and this may be the mechanism underlying the regulation of anxiety by CB1Rs in the amygdala of NHPs.

Motor assessment of developing common marmosets

Motor development has been extensively studied in human infants and children, with several established scales for the evaluation of motor functions. However, the study of the neuronal mechanisms underlying human motor development is hampered by the lack of good animal models. The common marmoset （Callithrix jacchus）, a small New World monkey, has recently attracted much attention as a potential non- human primate model for understanding human physiology and diseases. However, little is known about its gross motor development. In the present study, we found that marmosets have a critical period for motor development in postnatal weeks 2 to 5, and acquire most of their motor skills by 8 weeks of age. We also developed methods to assess their motor functions, which will be useful for the evaluation of motor performance in marmoset models of human diseases. In addition, we found that marmosets exhibit a ＂head-to-tail＂ sequence of motor development similar to that found in humans, further supporting the notion that they provide a good animal model for studying the neuronal mechanisms underlying human motor development.

A modified light-dark box test for the common marmoset

The common marmoset （Callithrix jacchus） has attracted extensive attention for use as a non-human primate model in biomedical research, especially in the study of neuropsychiatric disorders. However, behavioral test methods are still limited in the field of marmoset research. The light-dark box is widely used for the evaluation of anxiety in rodents, but little is known about light-dark preference in marmosets. Here, we modified the light-dark test to study this behavior. The modified apparatus consisted of three compartments： one transparent open area and two closed opaque compartments. The closed compartments could be dark or light. We found that both adult and young marmosets liked to explore the open area, but the young animals showed more interest than adults. Furthermore, when one of the closed compartments was light and the other dark, the adult marmosets showed a preference for the dark compartment, but the young animals had no preference. These results suggest that the exploratory behavior and the light-dark preference in marmosets are age-dependent. Our study provides a new method to study exploration, anxiety, and fear in marmosets.

甲型肝炎灭活疫苗免疫原性的猴体实验

甲型肝炎病毒TZ84毒株经 2BS二倍体细胞培养制备成甲型肝炎灭活疫苗。采用普通狨猴和恒河猴进行疫苗的保护性研究。结果均有较好的抗体反应 ,而且对甲型肝炎病毒鲁兴株 (强毒株 )的攻击亦均有保护作用。表明该疫苗有较好的免疫效果。

甲型肝炎病毒动物模型研究

ＨＡＶ合—５２野毒株感染恒河猴和普通狨猴的结果表明，后者的易感性显然高于前者。用不同ＨＡＶ株实验性接种普通狨猴，结果显示均有特异性抗ＨＡＶ产生和粪便ＨＡＶ颗粒排出。接种Ｈ２株疫苗的狨猴无酶活性升高以及肝组织病理学改变，而接种不同野毒株者均有生化反应及肝脏病变。普通狨猴可成为ＨＡＶ研究的良好的动物模型。

甲型肝炎灭活疫苗接种普通狨猴的安全性和免疫原性

目的观察本实验室研制的甲型肝炎灭活疫苗的安全性和免疫原性。方法甲型肝炎病毒吕－８ 株在人胚肺二倍体细胞（ＫＭＢ１７）中增殖，收获的病毒液用福尔马林灭活制成实验性疫苗，接种普通狨猴。结果有 特异性抗ＨＡＶ产生，无血清酶活性升高和肝组织病理学改变。接种疫苗的狨猴均能抵抗甲肝病毒强毒株的攻击， 而对照组均出现酶活性升高和肝组织病理学改变。结论该疫苗具有良好的免疫原性和可靠的安全性。

一株引起普通棉耳狨猴死亡病毒的分离与鉴定

1999年6月,天津医科大学动物中心饲养的珍贵灵长类实验动物——普通棉耳狨猴群体中暴发急性呼吸道传染病,病死率高达33％。在排除细菌感染的基础上,通过死亡狨猴肺组织匀浆接种鸡胚和MDCK细胞的分离培养,分离出一株具有高血凝效价的病毒株。经双份血清试验及动物接种试验,确认该病毒是本次疾病流行的病原体。又进一步通过与常见呼吸道病毒标准毒株及血清进行交叉血凝抑制试验、电镜观察、RT-PCR技术并结合生物信息学方法对该毒株进行鉴定,确认本次疾病流行的病原体是副流感1型病毒中的仙台病毒。

绒猴口服200mg碘油有效供碘时间及药代动力学研究

为了探讨目前国内广泛使用的２００ｍｇ小剂量碘油口服后的药代动力学和代谢规律，我们选择了灵长目动物绒猴为实验对象，以药代动力学方法，对尿碘、粪碘、血碘、ＴＳＨ、Ｔ３、Ｔ４等观测指标，进行了严格的观察。结果证明：绒猴在口服该剂量碘油后，吸收迅速，生物利用率高，其主要排泄途径是尿液。其排泄方式为二房室模型（具有快相和慢相）；碘在体内的贮存时间为１８０～２７０天（６～９个月）。

腺相关病毒介导的狨猴P53基因沉默

目的在细胞和整体动物水平,利用RNA干扰技术下调绒猴p53基因表达。方法对狨猴p53基因做生物信息学分析,针对靶序列设计shRNA干扰序列,构建在腺相关病毒载体上,转染非洲绿猴肾细胞(cos-7),在细胞水平用荧光定量PCR检测p53mRNA抑制效果,以Western blot方法检测p53蛋白水平表达变化;优选shRNA干扰序列,包装含shRNA干扰序列的8型腺相关病毒,静脉注射感染狨猴;手术取少量肝脏组织,用Western blot和免疫组化方法检测p53蛋白水平的变化。结果细胞水平研究发现2个有效RNA干扰靶点,mRNA干扰效率分别为(82.7±8.1)%和(80.7±7.5)%(P<0.05);蛋白表达下调(77.3±11.5)%和(73.7±10.7)%(P<0.05);2只绒猴感染病毒后,经活体荧光成像分析可见病毒在肝脏、睾丸、颈部等位置分布,狨猴肝脏P53蛋白经Western blot、免疫组化分析未见明显变化。结论本研究在细胞水平实现绒猴P53基因表达下调,但整体动物水平狨猴肝脏P53蛋白表达未发现明显变化;今后需在感染方式等方面做进一步优化。

甲型肝炎病毒(HAV)感染普通狨猴的病理学观察

观察使用ＨＡＶ疫苗株和野毒株感染普通狨猴后引起的病理变化，并对病变作分级描述。结果显示，疫苗株感染的狨猴未见异常。

副粘病毒Tianjin株NP、P、M及L蛋白的生物信息学分析

为了进一步明确副粘病毒Tianjin株的来源和种系进化地位,探讨其高致病性的机制。对Tianjin株NP、P、M及L蛋白进行了生物信息学分析。进化树显示：Tianjin株属于副粘病毒亚科呼吸道病毒属,且很可能为仙台病毒新的基因型。相似性比较表明,P蛋白变异最大,相似性仅为78.7%～91.9%;L蛋白相似性最高,为96.0%～98.0%。序列比对显示：NP蛋白氨基酸序列中存在15个独特的变异位点,P蛋白存在29个,M蛋白存在6个,L蛋白存在29个。这些独特变异位点的存在很可能是导致Tianjin株在宿主来源和致病特点等方面与已知仙台病毒株具有较大差异的原因。

普通棉耳狨猴中乙型流感病毒的分离和鉴定

乙型流感病毒是引起人类局部流行性感冒的重要病原体 ,其起源和自然储存宿主目前仍不清楚 .1 999年夏季在某动物中心饲养的普通棉耳狨猴 ( Callithrix jacchus)群体中爆发了以呼吸道症状为主的急性传染病 ,死亡比率高达 1 /3.通过对死亡狨猴肺组织匀浆接种鸡胚和 MDCK细胞的分离培养 ,分离出病毒 .双份血清的红细胞凝集抑制试验证实 ,此分离株为此次狨猴群体感染流行的病原体 .通过甲型和乙型流感病毒标准血清鉴定 ,证实该分离株为乙型流感病毒 ,命名为 B/marmoset/China/1

普通狨猴的血清酶学分析

目的 探讨实用有效的普通狨猴血清酶学检验方法。方法 常规法分离血清,采用2,4-二硝基苯腙比色法,24 h内测定狨猴血清中异柠檬酸脱氢酶(ICD)活力,IFCC法测定丙氨酸转氨酶(ALT)、草氨酸转氨酶(AST)及γ-谷氨酰转肽酶(γ-GT)的活力,平板琼脂糖凝胶电泳测定乳酸脱氢酶(LDH)同工酶的活力水平,并与国内外相关实验结果进行比较。结果 75只普通狨猴的ALT、AST、γ-CT和ICD活力水平与国内外相关实验结果基本相符,不同试剂等实验条件会造成实验结果出现较大差异。结论 ALT、AST和ICD是甲型肝炎研究中狨猴肝脏损伤的有效指标。采用动物实验前自身对照值是可靠和科学的血清酶学分析方法。

普通棉耳狨猴副粘病毒自然感染的研究

对一个狨猴实验室中暴发急性呼吸道疾病进行了病因调查。通过动物剖检、病毒分离、电子显微镜检查和血凝及血凝抑制试验和动物感染实验 ,确定其病原是副流感病毒 型(仙台病毒 )。从而确定动物死亡原因是病毒引起肺炎感染造成。