封面图片说明:中国实验动物学领域的又一突破——建立携带人类自闭症基因的非人灵长类动物模型

 自闭症,也称孤独症,是一类多发于青少年的发育性神经精神疾病,目前的神经基础还未能查清,也没有效的药物治疗方法。2016年1月26日,中科院上海生命科学研究院神经科学研究所仇子龙研究组在《Nature》在线发表了《Autism-like behaviours and germline transmission in transgenic monkeys overexpressing MeCP2》（MECP2转基因猴的类自闭症行为表征与种系传递）一文,通过构建携带人类自闭症基因MECP2（第二个与甲基化DNA结合的蛋白质）的转基因猴模型及对/MECP2转基因猴进行分子遗传学与行为学分析,发现/MECP2转基因猴表现出类人类自闭症的刻板行为与社交障碍等行为。     

中国实验动物学领域的又一突破——建立携带人类自闭症基因的非人灵长类动物模型

<正>自闭症,也称孤独症,是一类多发于青少年的发育性神经精神疾病,目前的神经基础还未能查清,也没有效的药物治疗方法。2016年1月26日,中科院上海生命科学研究院神经科学研究所仇子龙研究组在《Nature》在线发表了《Autism-like behaviours and germline transmission in transgenic monkeys overexpressing MeCP2》(MECP2转基因猴的类自闭症行为表征与种系传递)一文,通过构建携带人类自闭症基因MECP2(第二个与甲基     

Spexin转基因小鼠的构建及表型初步分析

Spexin是一种具有多种生物学功能的神经肽,在代谢疾病及神经系统疾病中发挥重要作用.为了深入研究spexin的生理功能和相关机制,构建了spexin转基因小鼠,通过检测血液中spexin含量以及糖耐受实验,对模型小鼠进行了初步的表型分析.利用piggyBac(PB)转座子技术培育spexin转基因小鼠,采用聚合酶链反应(polymerase chain reaction,PCR)和酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)分别在基因层面和蛋白层面验证了模型动物是否构建成功.之后用饲喂高脂饲料的雄性小鼠进行糖耐受实验,对转基因模型小鼠进行表型分析.PCR结果显示,以靶向spexin的两对引物扩增,转基因型小鼠基因组DNA中能分别扩增出300 bp和301 bp的片段,而野生型小鼠则不能.ELISA结果中,转基因型小鼠血清中spexin含量较野生型小鼠显著升高;同一表型中,雌雄小鼠间血清中spexin水平无明显差异.饲喂高脂饲料85 d内,转基因型小鼠和野生型小鼠体重变化未观察到显著差异;糖耐受结果显示,转基因型小鼠每个时间点血糖浓度均低于野生型小鼠,且在15 min时具有显著性差异.结果显示成功构建了过表达spexin基因的小鼠模型.spexin表达水平的升高在一定程度上提高了C57BL/6小鼠的糖耐受能力.该模型为进一步研究spexin基因在动物体内的功能以及在代谢疾病发病机制中的作用奠定了基础.     

Pygo2转基因小鼠模型的建立及表型的初步分析

主要通过建立Pygo2转基因小鼠的模型对其表型进行初步分析.首先构建K14-2×flag-Pygo的转基因构件,经酶切、纯化后构建Pygo2转基因小鼠.出生后的仔鼠用PCR和Western方法检测基因型,并通过进一步的免疫组化验证Pygo2基因的表达.PCR检测获得7只转基因阳性鼠,6只Western检测为阳性.对转基因小鼠子代的胚胎和成体进行免疫组化证明,Pygo2基因在皮肤和乳腺组织中有过量表达.转基因小鼠的皮肤、乳腺以及鼠尾椎骨等组织出现了异常的表型.乳腺中有肿瘤组织的形成,且Pygo2在肿瘤中有大量表达.该模型的成功建立为进一步研究Pygo2的功能奠定了基础.     

<非洲绿猴和食蟹猴主要肠道细菌的实时定量 PCR 检测及分析

摘要:目的 非洲绿猴具有不同于食蟹猴和恒河猴等猕猴的生物学特征,如脂质代谢、自发性高血压、SIV 感染不引起获得性免疫缺陷综合症( AIDS)等,本研究拟对食蟹猴和非洲绿猴肠道细菌的相对含量进行比较研究。 方法随机选取 SIV 抗体阴性的成年食蟹猴和非洲绿猴各 12 只,排除肥胖和疾病个体,收集其新鲜粪便样本,按 200 mg每份等分后进行液氮速冻,-80 ℃ 保存。 其中 1 份进行肠道细菌的实时定量 PCR 分析。 结果 与食蟹猴相比,非洲绿猴双歧杆菌、乳酸菌以及肠杆菌相对含量显著偏低( P<0. 05) ,而普氏菌以及丁酸盐产生菌 ———普拉梭菌和真细菌相对含量更高( P<0. 05) 。 结论 非洲绿猴产丁酸盐的细菌相对含量更高,反映出其肠道免疫细胞稳态和黏膜完整性具有不同于食蟹猴的进化适应特征,为深入研究非洲绿猴对 SIV 感染不致病的相关机制奠定了基础。     

Tet-on系统诱导乳腺特异表达CUEDC2转基因小鼠的制备

目的：建立Tet-on系统诱导乳腺特异表达CUEDC2的转基因小鼠模型。方法：构建转基因表达载体,经细胞诱导表达验证后,酶切得到含有CUEDC2的线性DNA片段,并采用显微注射的方法及后续筛选鉴定,得到CUEDC2转基因小鼠。进而通过与乳腺特异表达的MMTV-rtTA转基因小鼠交配,得到CUEDC2/rtTA双阳性转基因小鼠。利用2mg/ml的多西环素（Doxycycline）诱导小鼠体内CUEDC2表达,通过Western Blot、免疫组化检测表达。结果：经诱导,CUEDC2/rtTA双阳性转基因小鼠的2个Founder的F1、F2代在转录水平和蛋白水平均成功表达CUEDC2, 并具有乳腺特异性。结论：Tet-on系统诱导乳腺特异表达CUEDC2的转基因小鼠制备成功.     

用慢病毒载体介导产生绿色荧光蛋白（GFP）转基因小鼠

以慢病毒（lentivirus）载体为骨架，携带绿色荧光蛋白（GFP）基因的假病毒，通过小鼠受精卵卵周隙注射，将其感染小鼠受精卵，经移植于假孕母鼠后获得转基因小鼠．应用PCR、荧光显微镜观察和流式细胞仪分析等技术，证明了GFP基因的整合率达到40%以上；实时定量PCR分析结果表明转基因小鼠中GFP基因整合的拷贝数约为40；染色体荧光原位杂交分析结果显示GFP基因在小鼠染色体上的整合是随机的，并通过交配可将外源基因遗传至子代，获得的多整合位点和不同表达水平的转基因小鼠具有明显的实际应用和研究价值．文中报道的慢病毒载体介导的转基因技术为高效制备和选育高表达转基因小鼠品系提供了一种有效的途径．     

国外期刊亮点

正中国科学家获得世界首例经过基因靶向修饰的小猴中国云南中科灵长类生物医学重点实验室YYuuyyuu NNiiuu等运用转基因技术成功实现了灵长类动物的特定基因的定点修饰,诞生了世界首只经过基因靶向修饰的小猴。研究成果发表于2014年2月13日出版的Cell。研究小组运用转录激活因子样效应物核酸酶(TALENs)和规律成簇的间隔短回文重复序列(CRISPR/CAS9)技术,实现了猕猴、食蟹猴2个物种的靶向基因修饰,诞生了世界首只经过基因靶向修饰的小猴。这是TALENs和CRISPR/CAS9这2项技术被首次证明在灵长类动物中的有效应用。研究小组还运用TALENs技术,实现猕猴、食蟹猴2个物种的基因靶向修饰的其他系列研究成果。     

华东地区人工饲养食蟹猴血液常规和血清生化值的测定

目的建立单笼饲养食蟹猴血液常规和血清生化的参考值,了解检测指标的性别差异。方法选取254只3～5岁健康食蟹猴,雄性84只,雌性170只,在清醒状态下采血。采用全自动血细胞分析仪和全自动生化分析仪检测血液常规和生化指标,并比较不同性别组间的差异。结果经统计分析,获取了单笼饲养食蟹猴血液常规和血清生化的参考值,雌雄食蟹猴血常规指标比较,RBC、HGB、MCHC、HCT、MONO、RDW-CV、MPV、PCT、P-LCR、PDW存在极显著性差异（P〈0.01）,BASO、NEUT%、LYMPH%、MCH、RDW-SD存在显著性差异（P〈0.05）,雌雄血生化指标比较,ALT、AST、ALP存在极显著性差异（P〈0.01）。结论建立了3～5岁食蟹猴血液常规和血清生化的参考值,同时表明部分指标存在性别差异。     

转基因小鼠乳腺表达人乳过氧化物酶的初步研究

从人基因组PAC文库中筛选出人乳过氧化物酶（hLPO）基因,利用长片段PCR的方法获得hLPO基因5’端约3kb片段,通过酶切方法获得hLPO基因3’端约27kb片段,将这两部分拼接并克隆到乳腺特异性表达载体pBC1上,构建以山羊β-casein启动区指导的hLPO的转基因表达载体pBC1-hLPO。利用显微注射的方法获得28只FO代小鼠,经PCR检测和Southerm杂交分析证实,有5只小鼠（4♂,1♀）为整合hLPO基因的转基因阳性小鼠,整合率为17.86％,整合拷贝数在1至5之间。利用SDS-PAGE凝胶电泳和Western blot印迹分析FO、F1代共3只雌性转基因小鼠乳样,结果表明hLPO重组蛋白的特异条带不明显。     

Towards a transgenic model of Huntington's disease in a non-human primate

Non-human primates are valuable for modelling human disorders and for developing therapeutic strategies; however, little work has been reported in establishing transgenic non-human primate models of human diseases. Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor impairment, cognitive deterioration and psychiatric disturbances followed by death within 10-15 years of the onset of the symptoms. HD is caused by the expansion of cytosine-adenine-guanine (CAG, translated into glutamine) trinucleotide repeats in the first exon of the human huntingtin (HTT) gene. Mutant HTT with expanded polyglutamine (polyQ) is widely expressed in the brain and peripheral tissues, but causes selective neurodegeneration that is most prominent in the striatum and cortex of the brain. Although rodent models of HD have been developed, these models do not satisfactorily parallel the brain changes and behavioural features observed in HD patients. Because of the close physiological, neurological and genetic similarities between humans and higher primates, monkeys can serve as very useful models for understanding human physiology and diseases. Here we report our progress in developing a transgenic model of HD in a rhesus macaque that expresses polyglutamine-expanded HTT. Hallmark features of HD, including nuclear inclusions and neuropil aggregates, were observed in the brains of the HD transgenic monkeys. Additionally, the transgenic monkeys showed important clinical features of HD, including dystonia and chorea. A transgenic HD monkey model may open the way to understanding the underlying biology of HD better, and to the development of potential therapies. Moreover, our data suggest that it will be feasible to generate valuable non-human primate models of HD and possibly other human genetic diseases.     

Autistic-like behaviours and hyperactivity in mice lacking ProSAP1/Shank2

Autism spectrum disorders comprise a range of neurodevelopmental disorders characterized by deficits in social interaction and communication, and by repetitive behaviour. Mutations in synaptic proteins such as neuroligins, neurexins, GKAPs/SAPAPs and ProSAPs/Shanks were identified in patients with autism spectrum disorder, but the causative mechanisms remain largely unknown. ProSAPs/Shanks build large homo- and heteromeric protein complexes at excitatory synapses and organize the complex protein machinery of the postsynaptic density in a laminar fashion. Here we demonstrate that genetic deletion of ProSAP1/Shank2 results in an early, brain-region-specific upregulation of ionotropic glutamate receptors at the synapse and increased levels of ProSAP2/Shank3. Moreover, ProSAP1/Shank2(-/-) mutants exhibit fewer dendritic spines and show reduced basal synaptic transmission, a reduced frequency of miniature excitatory postsynaptic currents and enhanced N-methyl-d-aspartate receptor-mediated excitatory currents at the physiological level. Mutants are extremely hyperactive and display profound autistic-like behavioural alterations including repetitive grooming as well as abnormalities in vocal and social behaviours. By comparing the data on ProSAP1/Shank2(-/-) mutants with ProSAP2/Shank3αβ(-/-) mice, we show that different abnormalities in synaptic glutamate receptor expression can cause alterations in social interactions and communication. Accordingly, we propose that appropriate therapies for autism spectrum disorders are to be carefully matched to the underlying synaptopathic phenotype.     

Shank3 mutant mice display autistic-like behaviours and striatal dysfunction

Autism spectrum disorders (ASDs) comprise a range of disorders that share a core of neurobehavioural deficits characterized by widespread abnormalities in social interactions, deficits in communication as well as restricted interests and repetitive behaviours. The neurological basis and circuitry mechanisms underlying these abnormal behaviours are poorly understood. SHANK3 is a postsynaptic protein, whose disruption at the genetic level is thought to be responsible for the development of 22q13 deletion syndrome (Phelan-McDermid syndrome) and other non-syndromic ASDs. Here we show that mice with Shank3 gene deletions exhibit self-injurious repetitive grooming and deficits in social interaction. Cellular, electrophysiological and biochemical analyses uncovered defects at striatal synapses and cortico-striatal circuits in Shank3 mutant mice. Our findings demonstrate a critical role for SHANK3 in the normal development of neuronal connectivity and establish causality between a disruption in the Shank3 gene and the genesis of autistic-like behaviours in mice.     

Site-independent expression of the chicken beta A-globin gene in transgenic mice

The level of expression of exogenous genes carried by transgenic mice typically varies from mouse to mouse and can be quite low. This behaviour is attributed to the influence of the mouse chromatin near the site of transgene integration. This 'position effect' has been seen in transgenic mice carrying the human beta-globin gene. It was however, abolished when DNase I hypersensitive sites (normally found 65 to 44 kilobases (kb) upstream) were linked to the human beta-globin transgene. Thus, the upstream DNA (previously named a dominant control or locus activation region, now denoted a locus control region) conferred the ability to express human beta-globin at high levels dependent on copy number on every mouse carrying the construct. We report here an investigation of chicken beta A-globin gene expression in transgenic mice. A 4.5-kb fragment carrying the beta A-globin gene and its downstream enhancer, without any far upstream elements, is sufficient to ensure that every transgenic mouse expresses chicken globin messenger RNA at levels proportional to the transgene copy number. Thus the chicken DNA elements that allow position-independent expression can function in mice. In marked contrast to the human beta cluster, these elements are no farther than 2 kb from the gene. The location of the elements within the cluster demonstrates that position independence can be mediated by DNA that does not define a gene cluster boundary.     

Increased affiliative response to vasopressin in mice expressing the V1a receptor from a monogamous vole.

Arginine vasopressin influences male reproductive and social behaviours in several vertebrate taxa through its actions at the V1a receptor in the brain. The neuroanatomical distribution of vasopressin V1a receptors varies greatly between species with different forms of social organization. Here we show that centrally administered arginine vasopressin increases affiliative behaviour in the highly social, monogamous prairie vole, but not in the relatively asocial, promiscuous montane vole. Molecular analyses indicate that gene duplication and/or changes in promoter structure of the prairie vole receptor gene may contribute to the species differences in vasopressin-receptor expression. We further show that mice that are transgenic for the prairie vole receptor gene have a neuroanatomical pattern of receptor binding that is similar to that of the prairie vole, and exhibit increased affiliative behaviour after injection with arginine vasopressin. These data indicate that the pattern of V1a-receptor gene expression in the brain may be functionally associated with species-typical social behaviours in male vertebrates.     

植物转基因沉默的发生机理和应用

随着转基因技术在农业生产等领域的深入发展,转基因沉默现象已引起越来越多人的关注.基因沉默的机制是多方面的,包括转基因的拷贝数和构型、在植物上的整合位点、转基因的转录水平等.研究基因沉默的原因并寻找相应的方法来进行控制,对于植物基因工程的发展以及转基因沉默的应用有着重要的意义.