The Development of a Novel Form of Mouse Cage Enrichment

This article describes the design and testing of a novel form of mouse cage enrichment. A cage insert was designed and developed to fulfill a number of enrichment goals pertaining to its effectiveness and practicality (i.e., to improve the environment of mice in laboratories while causing the minimum of interference to the husbandry and care procedures carried out by animal care staff). Behavioral and physical parameters were used over an 8-week period to assess effectiveness, with subjective opinions of cage cleaning staff and physical examinations of the inserts after cleaning being used to assess practicality. Results show that the insert was both a practical and effective form of mouse enrichment which produced a number of enriching effects without adversely affecting the animals, their environment, or the husbandry and care procedures.     

The Use of Cage Enrichment to Reduce Male Mouse Aggression

The complete cleaning of cages has been shown to reduce the level of intermale aggression in mice. This study investigated the effects of the addition of enrichment objects on postcage-cleaning aggression in male BALB/c mice. Enrichment objects were found to significantly reduce aggressive interactions during this period for up to 7 weeks and can make an overall economic saving to husbandry costs.     

The Use of Cage Enrichment to Reduce Male Mouse Aggression

The complete cleaning of cages has been shown to reduce the level of intermale aggression in mice. This study investigated the effects of the addition of enrichment objects on postcage-cleaning aggression in male BALB/c mice. Enrichment objects were found to significantly reduce aggressive interactions during this period for up to 7 weeks and can make an overall economic saving to husbandry costs.     

与小鼠胚胎发育相关新基因0610038D11Rik的研究

目的:初步分析与小鼠胚胎发育相关的新基因0610038D11Rik表达模式及生物学功能.方法:采用RT-PCR,全胚胎原位杂交和Northern Blotting技术对该基因进行表达谱分析;细胞免疫染色对其进行细胞结构定位.结果:全胚胎原位杂交结果显示0610038D11Rik在胚胎E9.5的端脑、间脑、菱脑和听泡处有较强的信号.随着神经管逐渐关闭,胚胎E10.5在背部神经嵴,神经管区也出现表达信号.E11.5时除了在上述部位表达外,心脏部位也检测到较弱的信号.RT-PCR和Northern Blot实验发现该基因在小鼠胚胎发育直至出生后均有持续性分布,并且在发育中后期的脑、心脏、肺、肾、肝脏,肌肉和舌等多种重要脏器广泛表达.细胞定位表明其主要集中在核内和细胞质中.结论:0610038D11Rik基因在小鼠的脑神经系统和多器官表达,提示该新基因可能在这些组织的发育过程中发挥重要的作用.     

The Novel Use of a Heterozygous Knockout Mouse for Embryofetal Development Assessment of a Glucokinase Activator

Glucokinase activators (GKAs), such as AZD1656, are designed as antihyperglycemic agents for diabetics and can cause dose‐limiting hypoglycemia in normal animals used in embryofetal development studies. Genetically modified heterozygous GK knockout (gk^del/wt) mice are less susceptible to severe GKA‐induced hypoglycemia than wild‐type mice due to their elevated baseline glucose levels. In this study, the gk^del/wt mouse was used as an alternative rodent strain for embryofetal development studies with AZD1656. Heterozygous global knockout gk^del/wt females were dosed with 20, 50, or 130 mg/kg/day of AZD1656 or vehicle for a minimum of 14 consecutive days before mating with wild‐type males and throughout organogenesis. Maternal effects were confined to slightly reduced food consumption, reduced body weight gain, and the pharmacologic effect of decreased plasma glucose. Fetuses were genotyped. Fetal weights at the high dose were slightly reduced but there was no effect on fetal survival. There were two specific major malformations, omphalocele and right‐sided aortic arch, with increased fetal incidence in mid‐ and high‐dose fetuses (e.g., omphalocele fetal incidence of 0.6, 0.7, 4.6, and 2% across the dose groups) plus increased incidences of minor abnormalities and variants indicative of either delayed or disturbed development. Fetal weight and abnormalities were unaffected by fetal genotype. The fetal effects are considered hypoglycemia related. There was no effect on embryofetal survival in the gk^del/wt mouse at AZD1656 exposures, which were 70× higher than those causing 75% fetal death in rabbits. This illustrates the value of genetically modified animals in unraveling target versus chemistry‐related effects.     

采取过滤网上的灰尘检测小鼠肝炎病毒的新方法的研究--基因检出法的应用

采用PCR法对不同样品进行检测 ,其目的是寻求在小鼠肝炎病毒 (MHV)感染早期 ,可以尽快检测出MHV的一种最佳检测材料。将被感染小鼠的新鲜粪便、干燥粪便、笼具过滤网上的尘埃以及病毒液作为基因检测样品 ,进行检测并比较检出的灵敏度。将灵敏度量化 ,以稀释成不同浓度的cDNA用巢式PCR法能够检出的稀释界限的倍数表示。其结果 :第一 ,从过滤网中的尘埃所得到的cDNA是新鲜粪便的 10 0倍 ;第二 ,从粪便检出的灵敏度不以粪便的干燥而发生变化。第三 ,如果将病毒液放在室温下 ,会随室温干燥 ,检出的灵敏度会大幅度的降低。从以上结果看 ,在过滤网上的尘埃中有大量的MHV存在 ,尘埃中的一部分病毒 ,即使放在室温也能够以稳定状态存在。作者认为 ,将饲育装置的排气过滤器中粘着的尘埃收集 ,作为检测的样品 ,结合巢式PCR法进行测定 ,对于频繁地检测单个饲育笼具或单元式饲育室内的MHV比较简便易行 ,是一种较好的早期监测感染鼠肝炎病毒的方法。     

A Novel Method for the Simultaneous Enrichment,Identification, and Quantification of Phosphopeptides and Sialylated Glycopeptides Applied to a Temporal Profile of Mouse Brain Development

We describe a method that combines an optimized titanium dioxide protocol and hydrophilic interaction liquid chromatography to simultaneously enrich, identify and quantify phosphopeptides and formerly N-linked sialylated glycopeptides to monitor changes associated with cell signaling during mouse brain development. We initially applied the method to enriched membrane fractions from HeLa cells, which allowed the identification of 4468 unique phosphopeptides and 1809 formerly N-linked sialylated glycopeptides. We subsequently combined the method with isobaric tagging for relative quantification to compare changes in phosphopeptide and formerly N-linked sialylated glycopeptide abundance in the developing mouse brain. A total of 7682 unique phosphopeptide sequences and 3246 unique formerly sialylated glycopeptides were identified. Moreover 669 phosphopeptides and 300 formerly N-sialylated glycopeptides differentially regulated during mouse brain development were detected. This strategy allowed us to reveal extensive changes in post-translational modifications from postnatal mice from day 0 until maturity at day 80. The results of this study confirm the role of sialylation in organ development and provide the first extensive global view of dynamic changes between N-linked sialylation and phosphorylation.The development of novel methods to simultaneously monitor multiple protein post-translational modifications (PTMs)¹ is an attractive tool for researchers. There is increasing evidence that both phosphorylation and glycosylation play important roles in cellular signaling networks during development and transformation of cells. Development of the mammalian brain is initiated during the embryonic stage and continues until adulthood. The brain originates through the proliferation of the telencephalon, the anterior part of the neural tube. Following differentiation, cells begin to migrate and associate into different brain structures. The brain structures are reorganized with the extension of axons and dendrites to communicate via synaptic terminal interactions (1, 2). These molecular interactions are governed by cell surface receptors that are often post-translationally modified with both N-linked glycans and phosphate groups, and studies have suggested that extracellular glycans play vital roles in the regulation of signal transduction pathways (3). For example, the myelin-associated glycoprotein (MAG) binds to cell surface glyco-conjugates GD1a, GT1b and Nogo receptors to form signaling complexes that inhibit axon outgrowth, whereas inhibition of Rho kinase reverses this process in a number of nerve cell types (4). There is growing evidence that both the differentiation and migration of neurons and the guidance of axons are regulated by sialic acid-containing glycoconjugates (5–7). Dietary supplementation of sialic acid leads to increases in sialic acid-containing glycoproteins in the frontal cortex and is associated with faster learning and memory in piglets (8). The nervous system contains an abundant array of sialylated molecules and it is therefore not surprising that changes in the sialiome (the content of sialylated glycoproteins (9)) of a neuron can regulate activity. Removal of sialic acids from membrane proteins by NEU3 in primary neurons leads to actin depolymerization and axonal growth through TrkA-mediated signaling (10). Moreover, the modulation of phosphorylation events because of changes in cell membrane sialylation has been described in cancer (11, 12). Tumors induced in sialyltransferase-deficient animals show altered expression of genes associated with focal adhesion signaling and display decreased phosphorylation of focal adhesion kinase, a target of β1-integrins (13). Sialylated glycoconjugates include N-linked glycans (attached to asparagine residues), O-linked glycans (attached to hydroxylated residues) and glycolipids. N-linked and O-linked glycans are predominantly processed through the endoplasmic reticulum and Golgi, and their protein targets are generally membrane associated, cell-surface or found in extracellular environments. Additional glycoconjugates include single sugar modifications such as O-linked N-acetylglucosamine, glycosaminoglycans, large lipopolysaccharides, and peptidoglycans.The ability to identify and quantify PTM in proteins using mass spectrometry (MS) relies on specific enrichment techniques to purify modified peptides-of-interest from among a complex mixture. As modified peptides are normally present in sub-stoichiometric levels compared with nonmodified peptides, they are generally not detected by MS without such specific enrichment. Many methods are available to enrich for single PTM, including phosphorylation and glycosylation. Titanium dioxide (TiO₂) chromatography was originally described for enrichment of phophopeptides from peptide mixtures using similar peptide loading conditions as used for immobilized metal affinity chromatography (14–16). However, using this procedure resulted in significant co-enrichment of nonphosphorylated peptides. Later we demonstrated that TiO₂ was able to selectively purify phosphorylated peptides and sialic acid-containing N-glycopeptides (9, 17) if peptide samples are loaded onto the TiO₂ resin in a buffer containing high organic solvent, very low pH and a multifunctional acid, such as 2,5- dihydroxybenzoic acid or glycolic acid.A recent study demonstrated the first simultaneous enrichment of N-glycopeptides and phosphopeptides from a complex peptide mixture (18). Peptides from mouse brain were separated using electrostatic repulsion hydrophilic interaction chromatography to identify 738 unique glycosylation sites representing 446 glycoproteins, and 915 unique phosphorylation sites from 382 phosphoproteins. This method however, required 3 mg of starting material and did not demonstrate the ability to selectively enrich sialylated glycopeptides from glycopeptides displaying neutral glycans. Furthermore, only a comparatively low number of phosphopeptides could be identified considering the generous protein load investigated. The method was also unable to separate deglycosylated peptides from phosphopeptides and no quantitative capabilities were shown.Here we report a novel multidimensional strategy that employs TiO₂ chromatography to enrich for sialylated glycopeptides and phosphopeptides followed by PNGase F treatment of the eluent and μHPLC hydrophilic interaction liquid chromatography (HILIC) to fractionate and separate formerly N-linked sialylated glycopeptides and phosphopeptides from complex membrane protein preparations of a variety of biological samples. The development of a quantitative N-linked sialiomics and phosphoproteomic strategy that is able to simultaneously monitor cell-(extracellular)-cell interactions and receptor signaling will be a valuable tool to study tissue development and cell stimulation.     

Enrichment from Birth Accelerates the Functional and Cellular Development of a Motor Control Area in the Mouse

Background

There is strong evidence that sensory experience in early life has a profound influence on the development of sensory circuits. Very little is known, however, about the role of experience in the early development of striatal networks which regulate both motor and cognitive function. To address this, we have investigated the influence of early environmental enrichment on motor development.

Methodology/Principal Findings

Mice were raised in standard or enriched housing from birth. For animals assessed as adults, half of the mice had their rearing condition reversed at weaning to enable the examination of the effects of pre- versus post-weaning enrichment. We found that exclusively pre-weaning enrichment significantly improved performance on the Morris water maze compared to non-enriched mice. The effects of early enrichment on the emergence of motor programs were assessed by performing behavioural tests at postnatal day 10. Enriched mice traversed a significantly larger region of the test arena in an open-field test and had improved swimming ability compared to non-enriched cohorts. A potential cellular correlate of these changes was investigated using Wisteria-floribunda agglutinin (WFA) staining to mark chondroitin-sulfate proteoglycans (CSPGs). We found that the previously reported transition of CSPG staining from striosome-associated clouds to matrix-associated perineuronal nets (PNNs) is accelerated in enriched mice.

Conclusions/Significance

This is the first demonstration that the early emergence of exploratory as well as coordinated movement is sensitive to experience. These behavioural changes are correlated with an acceleration of the emergence of striatal PNNs suggesting that they may consolidate the neural circuits underlying these behaviours. Finally, we confirm that pre-weaning experience can lead to life long changes in the learning ability of mice.     

mED2-一个小鼠胚胎发育的新基因

克隆参与胚胎发育的新基因并研究其表达规律和功能是揭示胚胎发育的基因调控机理的重要途径。囊胚形成和原肠形成是哺乳动物胚胎发育过程中的两个关键阶段。囊胚阶段发生了胚胎的第一次分化,是细胞多能性和分化的一个转折点。此时涉及的基因活动,既有维持胚胎干细胞全能性或多能性的基因活动,又有按照预定发育模式参与胚胎定向分化的基因活动。原肠期是胚胎发育过程中的第二个关键转折点,涉及到3个胚层的形成和细胞命运决定等多种变化。在这个时期胚胎获得了胎儿原基的所有信息,新组织的产生和细胞迁移的再生组织与形态发生、细胞增殖、细胞分化、模式形成等存在着非常复杂而相互协调的关联。大多数细胞正由原来的多潜能逐渐向寡潜能发展,控制组织器官形态建成的基因正逐渐开启。这两个时期的基因表达图式、特征和种类会有很大的差异和变化,因此研究这两个时期的新基因的表达规律和功能,将是了解胚胎发育的基因调控机理的重要途径。文章以这两个时期胚胎为原始材料,利用减法杂交方法克隆到一新的小鼠胚胎基因mED2,对其进行了表达规律和生物学功能的初步分析。RT-PCR-Southern和原位杂交实验表明,mED2基因转录水平具有发育阶段的依赖性;随着发育过程的进行,其表达主要在胚神经系统和中胚层衍生的组织表达。mED2基因活性的knockdown对于合子的卵裂和植入前早期胚胎发育均有抑制作用。亚细胞定位实验表明,mED2基因编码的蛋白基本定位于细胞核膜及其临近的内膜细胞器（粗糙内质网和高尔基体）。根据生物信息学分析,mED2蛋白可能为一跨膜蛋白且与含有硫氧还蛋白结构域的蛋白有部分匹配。由此推测mED2基因参与了小鼠植入前早期胚胎发育,其基因产物可能通过蛋白之间的相互作用,即对蛋白进行后期修饰、折叠及行使分子伴侣等作用来活化或抑制其靶蛋白的活性,进而参与小鼠的早期胚胎发育。     

The Shortest Isoform of Dystrophin (Dp40) Interacts with a Group of Presynaptic Proteins to Form a Presumptive Novel Complex in the Mouse Brain

Duchenne muscular dystrophy (DMD) causes cognitive impairment in one third of the patients, although the underlying mechanisms remain to be elucidated. Recent studies showed that mutations in the distal part of the dystrophin gene correlate well with the cognitive impairment in DMD patients, which is attributed to Dp71. The study on the expression of the shortest isoform, Dp40, has not been possible due to the lack of an isoform specific antibody. Dp40 has the same promoter as that found in Dp71 and lacks the normal C-terminal end of Dp427. In the present study, we have raised polyclonal antibody against the N-terminal sequence common to short isoforms of dystrophin, including Dp40, and investigated the expression pattern of Dp40 in the mouse brain. Affinity chromatography with this antibody and the consecutive LC-MS/MS analysis on the interacting proteins revealed that Dp40 was abundantly expressed in synaptic vesicles and interacted with a group of presynaptic proteins, including syntaxin1A and SNAP25, which are involved in exocytosis of synaptic vesicles in neurons. We thus suggest that Dp40 may form a novel protein complex and play a crucial role in presynaptic function. Further studies on these aspects of Dp40 function might provide more insight into the molecular mechanisms of cognitive impairment found in patients with DMD.     

Genetics of Mandible Form in the Mouse

The underlying determination of phenotypic variability and covariability is described for 14 traits that define the morphological size and shape of the mature mouse mandible. Variability is partitioned into components due to direct additive and dominance genetic effects, indirect maternal additive genetic effects, genetic covariance between direct additive and indirect maternal additive effects and common and residual environmental effects. Multivariate analyses of the dimensionality of genetic variability indicate several complex and independent genetic components underlie the morphological form of the mandible. The multidimensional nature of the genetic components suggests a complex picture with regard to the consequences of selection on mandibular form.     

Neuroprotection in a Novel Mouse Model of Multiple Sclerosis

Multiple sclerosis is an immune-mediated, demyelinating and neurodegenerative disease that currently lacks any neuroprotective treatments. Innovative neuroprotective trial designs are required to hasten the translational process of drug development. An ideal target to monitor the efficacy of strategies aimed at treating multiple sclerosis is the visual system, which is the most accessible part of the human central nervous system. A novel C57BL/6 mouse line was generated that expressed transgenes for a myelin oligodendrocyte glycoprotein-specific T cell receptor and a retinal ganglion cell restricted-Thy1 promoter-controlled cyan fluorescent protein. This model develops spontaneous or induced optic neuritis, in the absence of paralytic disease normally associated with most rodent autoimmune models of multiple sclerosis. Demyelination and neurodegeneration could be monitored longitudinally in the living animal using electrophysiology, visual sensitivity, confocal scanning laser ophthalmoscopy and optical coherence tomography all of which are relevant to human trials. This model offers many advantages, from a 3Rs, economic and scientific perspective, over classical experimental autoimmune encephalomyelitis models that are associated with substantial suffering of animals. Optic neuritis in this model led to inflammatory damage of axons in the optic nerve and subsequent loss of retinal ganglion cells in the retina. This was inhibited by the systemic administration of a sodium channel blocker (oxcarbazepine) or intraocular treatment with siRNA targeting caspase-2. These novel approaches have relevance to the future treatment of neurodegeneration of MS, which has so far evaded treatment.     

在小鼠发育过程中线粒体基因组新型R环的表达

本实验室在成年小鼠线粒体DNA(mtDNA)D环上发现了一个新颖的轻链RNA转录本,这个RNA能够同DNA双链结合,形成一个稳定的DNA-RNA杂合结构(R环)。在此基础上,利用RT-PCR和Northern印迹法检测了小鼠线粒体基因组中R环的时空表达的特点。发现R环在小鼠不同组织、不同发育阶段中的表达水平有差异,其表达模式具有分化的位相性和时序性,提示R环有可能作为参与调控线粒体基因表达的分子,因而具有重要意义。     

小鼠基因组研究进展

小鼠基因组研究进展李善如１,２王冬平１陈永福２（１．军事医学科学院实验动物中心,北京１０００７１）（２．中国农业大学生物学院,北京１０００９４）ＴｈｅＤｅｖｅｌｏｐｍｅｎｔｏｆＭｏｕｓｅＧｅｎｏｍｅＲｅｓｅａｒｃｈＬＩＳｈａｎｒｕ１,２ＷＡＮＧＤｏｎ...     

Acanthamoeba-Campylobacter Coculture as a Novel Method for Enrichment of Campylobacter Species

In this study, we present a novel method to isolate and enrich low concentrations of Campylobacter pathogens. This method, Acanthamoeba-Campylobacter coculture (ACC), is based on the intracellular survival and multiplication of Campylobacter species in the free-living protozoan Acanthamoeba polyphaga. Four of the Campylobacter species relevant to humans and livestock, Campylobacter jejuni, C. coli, C. lari, and C. hyointestinalis, were effectively enriched by the coculture method, with growth rates comparable to those observed in other Campylobacter enrichment media. Studying six strains of C. jejuni isolated from different sources, we found that all of the strains could be enriched from an inoculum of fewer than 10 bacteria. The sensitivity of the ACC method was not negatively affected by the use of Campylobacter-selective antibiotics in the culture medium, but these were effective in suppressing the growth of seven different bacterial species added at a concentration of 10⁴ CFU/ml of each species as deliberate contamination. The ACC method has advantages over other enrichment methods as it is not dependent on a microaerobic milieu and does not require the use of blood or other oxygen-quenching agents. Our study found the ACC method to be a promising tool for the enrichment of Campylobacter species, particularly from water samples with low bacterial concentrations.     

小鼠一个新基因mLPTS的克隆、表达及亚细胞定位

利用EST拼接技术,RT－PCR及DNA序列测定,首次成功克隆了小鼠新基因mLPTS。获得的mLPTS基因片段长1244bp,编剧了一个由332个氨基酸组成的蛋白质。该蛋白质与人的LPTS蛋白有78％的同源性,LPTS基因是本实验室通过定位候选克隆策略获得的一个新的肝癌相关基因。它在肝癌组织中不表达或低表达,并参与细胞生长的负调控。小鼠mLPTS基因在小鼠的各个组织中都有表达,与人LPTS基因的表达组织分布相同。分析比较了LPTS蛋白在不同物种间的序列同源性,发现LPTS在进化上是高度保守的,是一个具有重要功能的基因。将mLPTS基因与绿色荧光蛋白EGFP融合构建真核表达载体,在中国仓鼠卵CHO细胞中表达,发现mLPTS基因表达产物位于细胞核仁中,为进一步研究该基因的功能及作用途径提供了重要信息。     

An Optimized Proteomics Approach Reveals Novel Alternative Proteins in Mouse Liver Development

Alternative ORFs (AltORFs) are unannotated sequences in genome that encode novel peptides or proteins named alternative proteins (AltProts). Although ribosome profiling and bioinformatics predict a large number of AltProts, mass spectrometry as the only direct way of identification is hampered by the short lengths and relative low abundance of AltProts. There is an urgent need for improvement of mass spectrometry methodologies for AltProt identification. Here, we report an approach based on size-exclusion chromatography for simultaneous enrichment and fractionation of AltProts from complex proteome. This method greatly simplifies the variance of AltProts discovery by enriching small proteins smaller than 40 kDa. In a systematic comparison between 10 methods, the approach we reported enabled the discovery of more AltProts with overall higher intensities, with less cost of time and effort compared to other workflows. We applied this approach to identify 89 novel AltProts from mouse liver, 39 of which were differentially expressed between embryonic and adult mice. During embryonic development, the upregulated AltProts were mainly involved in biological pathways on RNA splicing and processing, whereas the AltProts involved in metabolisms were more active in adult livers. Our study not only provides an effective approach for identifying AltProts but also novel AltProts that are potentially important in developmental biology.     

cDNA of a Novel mRNA Expressed Predominantly in Mouse Kidney

We examined embryonic carcinoma (EC) cells for a potential prototype molecule of C3, the third component of complement. PCR primers, corresponding to the base sequence derived from the C3 cDNA of several species, were used for PCR amplification of the EC cell cDNA. All the PCR products obtained had the same sequence and showed no sequence homology to C3. Subsequently, cDNA clones were isolated from a mouse liver cDNA library using the PCR product as a probe. Unexpectedly, neither the base sequence of the cDNA clones nor the amino acid sequence deduced from the cDNA showed homology to C3, although partial homology was observed to a number of sequences from EST databases. We designated this new clone NCU-G1. Northern hybridization experiments revealed that NCU-G1 is expressed constitutively not only in the mouse fetus but also in various mouse tissues, and is most abundant in the kidney cortex.     

Form of Selenium in Selenite Enrichment Media for Isolation of Salmonellae

Selenite-F and selenite-cystine media, commercially available for the routine isolation of salmonellae, were treated by anion exchange chromatography to separate the selenium from other components of the media. A chemical assay, based on an ascorbic acid reduction, showed that the selenium was all in the form of selenite.