MDMPR:小鼠发育代谢表型库

小鼠发育代谢表型库(Mouse Developmental and Metabolic Phenotype Repository,MDMPR)是一个致力于小鼠资源和表型数据实时共享的开放性平台,它依托于科技部重点研发计划“发育编程及其代谢调节”专项项目“建立小鼠发育代谢表型库”。该项目预计在5年内完成500个发育代谢相关小鼠敲除模型的建立,并对其表型数据进行标准化的解析、建立表型数据库。MDMPR作为一个资源及数据集成的库,由多个子系统作为支撑,包括ES细胞数据库、项目管理系统、繁育管理系统、精子库管理系统、表型分析系统,信息化管理深入到项目中每个环节,从基因突变ES细胞制备、基因突变小鼠制备、小鼠繁育,精子冻存到最终的表型分析、数据处理及展示,保证了MDMPR产生数据的真实性及实时性。MDMPR除了不断地推进项目进行,增加自身产生的数据外,也在积极的整合其他的资源及数据,如人特异性基因敲除ES细胞库、蛋白相互作用数据库(STRING)、核心转录调节环路(dbCoRc)和Enhancer-Indel数据库,今后还将进一步整合,帮助发育代谢及其他领域的研究人员能够一站式的获取所需资源和数据、加快研究进程,最终服务于全人类的医疗事业。     

小鼠心脏发育的形态学研究

目的建立小鼠心脏正常发育的时间表以及对应的形态学特征模式.方法小鼠胚胎ED8.5、ED9.5、ED10.5、ED11.5、ED12.5、ED14.5、ED16.5、ED18.5和P1(postnatal day)(出生后1 d的仔鼠)标本,进行整体或心脏部位不同轴向切片,HE染色,采用PCTV图像分析系统,对各时相小鼠心脏形态发育特征进行研究.结果 ⑴细胞结构发育的时空模式:① ED8.5时,生心板形成;ED9.5时,心肌细胞呈不规则的纺锤形,细胞的大小多样化,细胞核小;ED10.5时,小血管和着色较浅的肌原纤维出现,细胞之间连接较松;ED11.5时,心肌纤维排列较紧,纵断面上呈细长形,横断面上呈不规则多角形;ED12.5时,细胞核着色更清晰,心肌细胞形状逐渐规则,细胞之间紧密连接,同时闰盘结构出现在心室心肌细胞.②ED12.5时心肌小梁结构第一次在心室出现,ED14.5时增厚,而在心房少见心肌小梁.⑵心室结构的形成和心脏发育的成熟:①心肌间充质网络结构在ED10.5的心室中明显呈现,随着它的发育,心室的心内膜在ED11.5出现,心室心外膜可以辨认.②房室隔在ED12.5完全形成,心内膜垫在ED12.5开始发生并快速发育,促进室间隔在ED14.5完全形成.③心包膜在ED16.5可明显辨认,心包膜腔形成,此时近段流出道心内膜垫完全心肌细胞替换.结论肌原纤维细胞和心肌间充质细胞同时在ED10.5出现,提示肌原纤维对心肌细胞的成型和心肌化起作用.细胞的结构变化和心肌层的成熟过程,显示小鼠心脏部位成熟时间的不同,心室成熟相对较晚.     

组织特异RNAi转基因小鼠模型的构建

RNAi是一种行之有效的基因沉默的新方法,被广泛地应用于基因功能的研究、疾病的治疗以及新型疫苗的研制等领域.本研究通过原核显微注射干扰载体的方法制备转基因小鼠.选用皮肤组织特异表达的人源角蛋白14（K14）基因启动子（2000bp）作为表达载体启动子,成功地驱动融合表达载体EGFP-shRNA进行干扰片段前体的转录,进而生成成熟的干扰片段,靶向小鼠BMP4基因使其发生沉默.所得到的转基因小鼠及其杂交后代经PCR和Southern杂交鉴定,结果表明外源基因准确无误地整合到小鼠基因组.Northern杂交结果证明,小干扰RNA在皮肤组织中有较高水平的表达,在肺和肠组织中有较低水平的表达.研究结果表明,利用PolⅡ型（K14）启动子驱动shRNA融合转录本的表达,在特定组织高表达siRNA,从而达到抑制特定组织目的基因表达的技术路线是可行的.同时为利用K14启动子进行毛囊相关基因干扰研究积累了基础数据,为制备组织特异抑制基因表达的转基因大家畜提供了一个参考方法.     

Hr突变体转基因小鼠的构建和表型分析

无毛基因（Hr）定位于8p12,在染色体上跨越14 kb,包含19个外显子。无毛基因的自发突变能引起人和动物毛发脱落及相关毛发疾病的产生。为深入研究Hr基因的功能,本文利用Gateway技术构建Hr表达载体,在该基因的3 427位引入点突变(G→A),通过显微注射建立转基因小鼠。采用PCR方法鉴定出阳性的转基因小鼠,确定首建鼠,通过与C57BL/6小鼠回交后互交数代建系。观察转基因小鼠毛发生长发育规律。结果表明,成功构建了pRP(Exp)-EF1A>mHairless mutant>IRES/EGFP真核表达载体,通过与野生型小鼠杂交获得阳性子代,进行同窝交配,第2代小鼠出生后14 d开始脱发,30 d左右脱落的毛发重新长出。取部分皮肤组织做石蜡切片,皮肤组织学观察发现,脱毛期无毛小鼠毛囊瓦解,真皮内形成大小不等的包囊,毛发重新生长时,真皮内见大量新生的毛囊。蛋白印迹实验表明,转基因小鼠脱发时HR蛋白表达量明显高于同龄阴性小鼠。本文成功建立稳定遗传的Hr突变的转基因小鼠品系,推测无毛基因突变引发转基因小鼠的脱发,为研究Hr基因的功能提供了良好的动物模型。     

小鼠电融合四倍体胚胎早期发育的研究

多倍体发育现象在低等动物,尤其是在无脊椎动物中比较普遍,在哺乳动物中多倍体往往发育到胚胎早期就死亡,因而在自然界中不能存活。探讨哺乳类四倍体不能存活的原因是当今生物学研究的热点问题之一。目前,电融合技术是大量获得四倍体胚胎的主要手段。本以8－12周龄昆明小白鼠为实验对象,取其2－细胞胚胎于Whitten氏液中进行电融合,再移入CZB液中培养。电刺激的条件分别为：1．0kv/cm,40μs,和0．8kv/cm,80μs。对融合胚发育的状态、细胞数目及染色体组成等进行观察的结果表明,融合胚培养24小时后发育到4－细胞期,并发生致密化（Tab.1)。融合胚的囊胚形成时间与体内发育的同期胚胎及体外培养的同期二倍体胚胎基本一致（Tab,2;Fig.1)。融合囊胚的细胞数平均为13．0±4．95,显（P＜0．01）少于体外发育囊胚（37．0±5．92）和体内发育囊胚（41．6±2．4）的细胞数（Tab.3)。融合胚各个分裂相的染色体数目分布情况见Fig.2,其四倍体（4n＝80）率为42．86％,非整倍体率达57．14％。而对照组的体内发育囊胚的非整倍体率仅为11．54％;（Tab.4),与Kaufman的结果（12．4％）很接近,说明本实验中的染色体制备技术和分析方法是可行的。融合囊胚的细胞数目减少和高发的非整倍体率可能是四倍体胚胎发育能力差的原因。     

小鼠卵母细胞的冷冻—解冻及其体外发育的研究

试验比较四种防冻剂对小鼠核泡期(germinal vesicle stage)无卵丘细胞的卵母细胞(下称裸卵),在各种不同条件下冷冻-解冻的影响,并用乙二醇为防冻剂,冷冻-解冻后,形态正常的裸卵1137枚,经体外培养排出第一极体、发育达中期Ⅱ的成熟率为43.2％(491/1137)。用体外获能的小鼠附睾尾精子进行体外受精的受精率为25.4％(31/122)。继续培养进一步发育至2-细胞期的发育率为17.3％(55/318)。对照组(新鲜裸卵)的成熟率为48.6％(158/325),受精率为26.4％(388/144)。两者无显著差异(P<0.05)。     

发育特异基因及其在RNA水平上的表达

从λ噬菌体gtll的cDNA表达文库筛选到7个胡萝卜体细胞发育特异的基因克隆,并对它们进行了纯化、分离噬菌体DNA,限制酶消化,琼脂糖凝胶电泳鉴定,以及将cDNA插入片段再克隆到pIBI或p^Uc18质粒等处理。然后,通过Southern blot分析,证明克隆16,22,50及60等为新的cDNA基因。此外,又通过Northern blot分析测定了这些基因的组织特异性表达及时序表达。结果表明,克隆22是一种受发育调节,与胡萝卜体细胞胚胎形成密切相关的基因。     

表型可塑性变异的生态-发育机制及其进化意义

表型可塑性赋予生物个体在不同环境条件下通过产生不同表型来维持其适合度的能力.研究结果显示多数可塑性变异的产生是基于对环境变异信号的响应、改变基因表达式样并调整发育轨迹的结果,表观遗传调控体系在基因选择性表达和可塑性变异的跨世代传递过程中发挥了重要作用.不同物种和种群对环境变化的敏感性、发生可塑性变异的能力以及可塑性反应模式不尽相同,预示着控制可塑性能力并独立于控制性状的可塑性基凶的存在,这些基因是直接响应环境信号并控制表型表达的调控基因.表型可塑性不仅是物种适应性进化的一个重要方面,也是选择进化的产物,物种的表型可塑性变异对其生态适应和进化模式有深远的影响.     

Prepulse inhibition （PPI） of tactile startle response in recombinant congenic strains of mice： QTL mapping and comparison with acoustic PPI

Prepulse inhibition (PPI) of the startle response is a psychophysiological measure of sensorimotor gating believed to be cross-modal between different sensory systems.We analyzed the tactile startle response (TSR) and PPI of TSR (tPPD,using light as a prepulse stimulus,in the mouse strains A/J and C57BL/6J and 36 recombinant congenic strains derived from them.Parental strains were significantly different for TSR,but were comparable for tPPI.Among the congenic strains,variation for TSR was significant in both genetic backgrounds,but that of tPPI was significant only for the C57BL/6J background.Provisional mapping for loci modulating TSR and tPPI was carded out.Using mapping data from our previous study on acoustic startle responses (ASR) and PPI of ASR (aPPI),no common markers for aPPI and tPPI were identified.However,some markers were significantly associated with both ASR and TSIL at least in one genetic background.These results indicate cross-modal genetic regulation for the startle response but not for PPI,in these mouse strains.     

Developmental expression of insulin-like growth factor II receptor (IGF-IIR) in congenic mouse embryonic lungs: Correlation between IGF-IIR mRNA and protein levels and heterochronic lung development

Embryonic lung maturation in the H-2 congenic pair, B10.A and B10, proceeds at different rates. The dependence of this heterochronic development on maternal haplotype suggests the involvement of a parentally imprinted gene. Since B10.A (H-2^b) and B10 (H-2^b) mice are genetically identical except for a 3-18 cM region of chromosome 17 that includes the H-2 complex, we sought a promising candidate gene(s) involved in regulating the rate of lung development from genes encoded in this region. The best candidate is the gene encoding the type II insulin-like growth factor receptor (IGF-IIR), whose ligand is the growth factor IGF-II. Only the maternal copy of this gene is expressed in postimplantation embryos. This receptor does not appear to transduce mitogenic signals; instead, IGF-IIR appears to regulate the levels of its ligand available to the growth-promoting type I IGF receptor (IGF-IR). Using in situ hybridization and indirect immunofluorescence, we demonstrate that IGF-IIR mRNA and protein are localized throughout the pulmonary mesenchyme, as well as in branching epithelia of the pseudoglandular and canalicular stages. We also examined the levels of IGF-IIR mRNA and protein expression by RNase protection assay and ligand blotting during the embryonic period of lung development in B10.A and B10 mice, and found that there is a highly significant positive correlation of IGF IIR levels with progressive development in both strains. Further, slower developing B10.A lungs contain significantly higher levels of IGF-IIR mRNA and protein than the more rapidly developing B10 lungs. These results suggest that haplotype-dependent elevation of IGF-IIR levels reduces the available concentration of IGF-II, resulting in a decreased rate of morphogenesis in B10.A mice. Heterochronic lung maturation, then, appears consequent to variable extracellular levels of this important growth factor. These results may be of clinical importance to predicting susceptibility to Respiratory Distress Syndrome in prenatal newborns. © Wiley-Liss, Inc.     

Mouse model for analysis of non-MHC genes that influence allogeneic response: recombinant congenic strains of OcB/Dem series that carry identical H2 locus

Alloreactivity is the strongest known primary immune response. Its clinical manifestations are graft rejection, graft-versus-host disease and graft-versus-leukemia effect. The strongest stimulation by allogeneic cells is due to incompatibility at the major histocompatibility complex (MHC) genes. However, the non-MHC genes also participate in allogeneic response. Here we present a mouse model for study of the role of non-MHC genes in regulation of alloreactivity and show that they besides encoding antigens also regulate the responsiveness. Recombinant congenic strains (RCS) of O20/A (O20)-c-B10.O20/Dem (OcB/Dem) series have been derived from the parental strains O20 and B10.O20, which carry identical MHC haplotypes (H2^pz) and therefore their differences in alloantigen response depend only on non-MHC genes. We have tested a MLR response by spleen cells of the strains O20, B10.O20, and 16 OcB/Dem strains through stimulation by cells from strains C57BL/10 (H2^b), BALB/c (H2^d), CBA (H2^k), and DBA/1 (H2^q) alloantigens. Proliferative response of O20, B10.O20 and OcB/Dem strains to these four alloantigens exhibited a similar but not completely identical pattern of reactivity. The responses to different alloantigens were highly correlated: C57BL/10-BALB/c r = 0.87, C57BL/10-CBA r = 0.84, C57BL/10-DBA/1 r = 0.83. Cluster analysis of the responses by O20, B10.O20, and OcB mice identified groups of strains with distinct patterns of response. This data shows that two main types of genes influence MLR: 1. structural genes for major and minor alloantigens and 2. genes regulating T-cell receptor signal transduction or mediating costimulatory signals by antigen-presenting cells.     

Applications of congenic strains in the mouse

The sequencing of the human and the mouse genomes has shown that the chromosomes of these two species contain approximately 30,000 genes. The biological systems that can be studied in an individual or in a tissue result from complex interactions within this multitude of genes. Before describing these interactions, it is necessary to understand the function of each gene. In the mouse, congenic strains are developed to introduce a chromosomal segment in a given inbred genetic background. One can then compare the biological effects of different alleles at the same locus in the same genetic background or the effect of a given allele in different genetic backgrounds. One can also introduce into different congenic strains with the same genetic background genes which control a complex genetic trait, then combine these genes by appropriate crosses to study their interactions. Although the chromosomal segment transferred into a congenic strain usually contains up to several hundreds of genes, molecular markers can be used to reduce this number as well as the number of crosses required for the development of congenic strains.     

Initial locomotor sensitivity to cocaine varies widely among inbred mouse strains

Initial sensitivity to psychostimulants can predict subsequent use and abuse in humans. Acute locomotor activation in response to psychostimulants is commonly used as an animal model of initial drug sensitivity and has been shown to have a substantial genetic component. Identifying the specific genetic differences that lead to phenotypic differences in initial drug sensitivity can advance our understanding of the processes that lead to addiction. Phenotyping inbred mouse strain panels are frequently used as a first step for studying the genetic architecture of complex traits. We assessed locomotor activation following a single, acute 20 mg/kg dose of cocaine (COC) in males from 45 inbred mouse strains and observed significant phenotypic variation across strains indicating a substantial genetic component. We also measured levels of COC, the active metabolite, norcocaine and the major inactive metabolite, benzoylecgonine, in plasma and brain in the same set of inbred strains. Pharmacokinetic (PK) and behavioral data were significantly correlated, but at a level that indicates that PK alone does not account for the behavioral differences observed across strains. Phenotypic data from this reference population of inbred strains can be utilized in studies aimed at examining the role of psychostimulant‐induced locomotor activation on drug reward and reinforcement and to test theories about addiction processes. Moreover, these data serve as a starting point for identifying genes that alter sensitivity to the locomotor stimulatory effects of COC.     

Genetic analysis of nonhistone chromosomal protein inheritance in recombinant inbred mouse strains using two-dimensional electrophoresis

Analysis of hepatic nonhistone chromosomal protein (NHCP) expression in male mice from progenitor strains (C3H/HeN, C57BL/6N), their F₁ hybrid (B6C3), and seven recombinant inbred strains (RIs) (B6N×C3N) by high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) detected 16 NHCPs whose expression in RIs could be correlated to each other and with strain distribution patterns (SDP) of 20 genetic markers differing in the progenitors. Of the 400₊ NHCP spots detected in RI 2D-PAGE maps, 172 were common to progenitors and all RIs. There was a characteristic absence of five NHCPs in one RI, Y. Ten C3H-specific and six C57-specific NHCP inherited in B6C3 also appeared in RIs. The SDP of C3H-specific NHCP 2 matched the SDP of beta-glucuronidase on chromosome 5 and carbonic anhydrase on chromosome 3, and C57-specific NHCP 5 SDP corresponded to that for nonagouti trait on chromosome 2. These 16 NHCP genetic marker inheritance differences detected in RIs add to the 23 previously established genetic marker differences between the progenitors.This study was supported in part by funds from NIH Grants CA 33305 and CA 16672 and Exxon Corporation, USA.     

Dissection of multigenic obesity traits in congenic mouse strains

Previous quantitative trait locus mapping (QTL) identified multigenic obesity (MOB) loci on mouse Chromosome (Chr) 2 that influence the interrelated phenotypes of obesity, insulin resistance, and dyslipidemia. To better localize and characterize the MOB locus, three congenic mouse strains were created. Overlapping genomic intervals from the lean CAST/Ei (CAST) strain were introgressed onto an obesity-susceptible C57BL/6 (BL6) background to create proximal (15 Mb–73 Mb), middle (63 Mb–165 Mb), and distal (83 Mb–182 Mb) congenic strains. The congenic strains showed differences in obesity, insulin, and lipid traits consistent with the original QTL analysis for the locus. Importantly, characterization of the MOB congenics localized the effects of genes that underlie obesity-related traits to an introgressed interval (73–83 Mb) unique to the middle MOB congenic. Conversely, significant differences between the lipid and insulin profiles of the middle and distal MOB congenics implicated the presence of at least two genes that underlie these traits. When fed an atherogenic diet, several traits associated with metabolic syndrome were observed in the distal MOB congenic, while alterations in plasma lipoproteins were observed in the middle MOB congenic strain.     

Genomic characterization of mutant laboratory mouse strains by exome sequencing and annotation lift-over

Background

Exome sequencing has become a popular method to evaluate undirected mutagenesis experiments in mice. However, the most suitable mouse strain for the biological model may be relatively distant from the standard mouse reference genome. For pinpointing causative variants, a matching reference with gene annotations is essential, but not always readily available.

Results

We present an approach that allows to use murine Ensembl annotations on alternative mouse strain assemblies. We resolved ENU-induced mutation screening for 8 phenotypic mutant lines generated on C3HeB/FeJ background aligning the sequences against the closely related, but not annotated reference of C3H/HeJ. Variants occurring in all strains were filtered out as specific for the C3HeB/FeJ strain but unrelated to mutagenesis. Variants occurring exclusively in all individuals of one mutant line and matching the inheritance model were selected as mutagenesis-related. These variants were annotated with gene and exon names lifted over from the standard murine reference mm9 to C3H/HeJ using megablast. For each mutant line, we could restrict the results to exonic variants in between 1 and 23 genes.

Conclusions

The presented method of exonic annotation lift-over proved to be a valuable tool in the search for mutagenesis-derived coding genomic variants and the assessment of genotype-phenotype relationships.     

Analysis of a lung defect in autophagy-deficient mouse strains

Yeast Atg1 initiates autophagy in response to nutrient limitation. The Ulk gene family encompasses the mammalian orthologs of yeast ATG1. We created mice deficient for both Ulk1 and Ulk2 and found that the mice die within 24 h of birth. When found alive, pups exhibited signs of respiratory distress. Histological sections of lungs of the Ulk1/2 DKO pups showed reduced airspaces with thickened septae. A similar defect was seen in Atg5-deficient pups as both Ulk1/2 DKO and Atg5 KO lungs show numerous glycogen-laden alveolar type II cells by electron microscopy, PAS staining, and increased levels of glycogen in lung homogenates. No abnormalities were noted in expression of genes encoding surfactant proteins but the ability to incorporate exogenous choline into phosphatidylcholine, the major phospholipid component of surfactant, was increased in comparison to controls. Despite this, there was a trend for total phospholipid levels in lung tissue to be lower in Ulk1/2 DKO and Atg5 KO compared with controls. Autophagy was abundant in lung epithelial cells from wild-type mice, but lacking in Atg5 KO and Ulk1/2 DKO mice at P1. Analysis of the autophagy signaling pathway showed the existence of a negative feedback loop between the ULK1 and 2 and MTORC1 and 2, in lung tissue. In the absence of autophagy, alveolar epithelial cells are unable to mobilize internal glycogen stores independently of surfactant maturation. Together, the data suggested that autophagy plays a vital role in lung structural maturation in support of perinatal adaptation to air breathing.     

Genetic basis for the psychostimulant effects of nicotine: a quantitative trait locus analysis in AcB/BcA recombinant congenic mice

Genetic differences in sensitivity to nicotine have been reported in both animals and humans. The present study utilized a novel methodology to map genes involved in regulating both the psychostimulant and depressant effects of nicotine in the AcB/BcA recombinant congenic strains (RCS) of mice. Locomotor activity was measured in a computerized open-field apparatus following subcutaneous administration of saline (days 1 and 2) or nicotine on day 3. The phenotypic measures obtained from this experimental design included total basal locomotor activity, as well as total nicotine activity, nicotine difference scores, nicotine percent change and nicotine regression residual scores. The results indicated that the C57BL/6J (B6) were insensitive to nicotine over the entire dose-response curve (0.1, 0.2, 0.4 and 0.8 mg/kg). However, the 0.8-mg/kg dose of nicotine produced a significant decrease in the locomotor activity in the A/J strain and a wide and continuous range of both locomotor excitation and depression among the AcB/BcA RCS. Single-locus association analysis in the AcB RCS identified quantitative trait loci (QTL) for the psychostimulant effects of nicotine on chromosomes 11, 12, 13, 14 and 17 and one QTL for nicotine-induced depression on chromosome 11. In the BcA RCS, nicotine-induced locomotor activation was associated with seven putative regions on chromosomes 2, 7, 8, 13, 14, 16 and 17. There were no overlapping QTL and no genetic correlations between saline- and nicotine-related phenotypes in the AcB/BcA RCS. A number of putative candidate genes were in proximity to regions identified with nicotine sensitivity, including the alpha2 subunit of the nicotinic acetylcholine receptor and the dopamine D3 receptor.