应用荧光原位杂交技术检测人βE珠蛋白基因在转基因小鼠染色体上的整合状态

为将荧光原位杂交技术应用于基因定位研究中,探讨一种能有效地检测转基因动物染色体上外源基因整合状态的实验方法,对小鼠腹腔注射秋水仙素后,取转基因小鼠骨髓制备中期染色体,将传统的FISH方法加以改进,检测外源基因在转基因小鼠染色体上的整合状态.检测结果表明,外源人βE珠蛋白基因已稳定地整合于小鼠染色体上.FISH能直观地反映外源基因在转基因动物染色体上的整合状态,该方法可对转基因动物及基因转移研究中的外源基因整合后进行染色体定位检测。 Abstract:To determine the integration site of human βE globin gene in the chromosomes of transgenic mice, transgenic mice carrying human βE globin gene were injected intraperitoneally with colchicines, then, bone marrow cells wereisolated and metaphase chromosomes were prepared, the traditional FISH method was improved to detect the integration site of humanβE globin gene in transgenic mice when combined with G-banding. Human t3E globin gene can bedetected in different position of different chromosomes in transgenic mice and FISH signals showed that two mice were heterozygous of human 13E globin gene and one was homozygous. Human t3E globin gene was integrated into thechromosomes of transgenic mice in a random pattern and the results demonstrated that FISH can be used to investigate the integration site of foreign genes in transgenic mice.     

应用荧光原位杂交技术检测人βE珠蛋白基因在转基因小鼠染色体上的整合状态

为将荧光原位杂交技术应用于基因定位研究中,探讨一种能有效地检测转基因动物染色体上外源基因整合状态的实验方法,对小鼠腹腔注射秋水仙素后,取转基因小鼠骨髓制备中期染色体,将传统的FISH方法加以改进,检测外源基因在转基因小鼠染色体上的整合状态.检测结果表明,外源人βE珠蛋白基因已稳定地整合于小鼠染色体上.FISH能直观地反映外源基因在转基因动物染色体上的整合状态,该方法可对转基因动物及基因转移研究中的外源基因整合后进行染色体定位检测。 Abstract:To determine the integration site of human βE globin gene in the chromosomes of transgenic mice, transgenic mice carrying human βE globin gene were injected intraperitoneally with colchicines, then, bone marrow cells wereisolated and metaphase chromosomes were prepared, the traditional FISH method was improved to detect the integration site of humanβE globin gene in transgenic mice when combined with G-banding. Human t3E globin gene can bedetected in different position of different chromosomes in transgenic mice and FISH signals showed that two mice were heterozygous of human 13E globin gene and one was homozygous. Human t3E globin gene was integrated into thechromosomes of transgenic mice in a random pattern and the results demonstrated that FISH can be used to investigate the integration site of foreign genes in transgenic mice.     

应用荧光原位杂交检测人凝血因子Ⅸ在转基因小鼠染色体上的整合

应用荧光原位杂交（FISH）技术检测两个转基因小鼠家系从F1到F4代的整合情况。阳性转基因小鼠98%~100%的中期分裂相,85%~94%的间期核出现杂交信号;阴性对照小鼠100%的中期分裂相、95%~96%的间期核未出现杂交信号。结果表明,该FISH实验条件能对转基因整合位点进行高效特异检测。本文分析的两家系转基因小鼠均为单位点整合, 但整合位点不同。各家系内F1到F4代的转基因小鼠均可检出整合染色体,且整合位点相同,表明外源基因稳定整合并遗传给后代。 Abstract:Fluorescence in situ hybridization （FISH） was used to detect the integration of hFⅨ on chromosomes of transgenic mice from F1 to F4 generation in two strains.For transgenic mice,98%~100% of metaphases and 85%~94% of interphases showed hybridization signal.For negative control mice,100% of metaphases and 95%~96% of interphases showed no hybridization signal.The results demonstrated that FISH developed to detect the integration sites of hFⅨ was high efficient and specific.The integration sites of the transgenic mice analyzed were both single but different between the two strains.The integration chromosomes can be found in the transgenic mice from F1 to F4 generation and the integration sites were the same as each of the strains,which indicated that the transgene was stably integrated and transmitted to offspring.     

基因组无痕修饰与动物转基因

动物转基因技术可以突破物种与年龄界限,有针对性地创造遗传变异.但是在转基因过程中,非目的序列的残留可能对后续研究造成障碍,甚至形成安全隐患.因此,对动物基因组进行无痕修饰的重要性日益凸显. 基因组无痕修饰目前主要有两套策略.一是人工核酸酶的非整合导入,这主要是通过RNA或蛋白质的形式实现,可有效避免核酸酶质粒DNA的残留.二是转基因载体的优化及整合后删除,它是借助Piggybac系统, 对末端重复单元之间的序列进行精确切离.本文总结了动物基因组无痕修饰的研究现状,并对其存在问题和应用前景做出评述,以期对我国的动物转基因研究和家畜转基因育种提供有益的参考和借鉴.     

生产转基因动物的技术现状

用实验手段,可将特定外源基因导入早期胚胎的细胞,由此整合到染色体上,还可通过被整合动物的生殖细胞系传给子代。这类整合外源基因的“新动物”被统称为转基因动物(Transgenic Animals)。 按人为意愿设计生产转基因动物,能达到如下若干目的:1)将理想的遗传物质导入动物染色体,以扩大种内遗传变异,迴避有性繁     

转基因动物整合位点的研究进展

转基因动物整合位点克隆及相关序列特征研究是探索外源基因整合机制和整合稳定性的重要手段。转基因整合位点序列的克隆方法主要有：个体基因组文库筛选法、反向PCR法、热不对称交互式PCR法和质粒回收法。4种方法各有优缺点,可根据不同条件选用。克隆到的整合位点序列表明,整合位点可能存在一定的共同特征。 Integration Sites of Transgenes in Transgenic Animals WU Bo,ZHU Zuo-Yan State Key Laboratory of Freshwater Ecology and Biotechnology,Institute of Hydrobiology, Chinese Academy of Science,Wuhan 430072,China Abstract:To study the mechanism of transgene ' s integration in the host genome,first step is to clone the integration-site sequence.The method employed for this purpose includes selection from genomic pool,IPCR,TAIL-PCR and plasmid rescued.Different method fits different cases depending on how the transgenics have been produced.The data of integration-site sequences showed some similar features existed. Key words：transgene; integration site; clone method     

通过染色体外同源重组建立乳腺中表达外源基因的转基因小鼠

为研究染色体外重组方法在创建转基因动物中的应用,选取牛asl酪蛋白基因的5′及3′侧翼区和氯霉素乙酰化酶编码区,构建了两个具有3 kb相互重叠的融合基因.将这两个DNA片段末端脱磷酸后,以摩尔比1:1的比例混合,通过显微注射导入小鼠受精原核,最后获得了11个品系的转基因小鼠.对其中10个品系的小鼠的整合分析表明,所注射的两个DNA片段均发生了染色体外同源重组,而且除了 1个品系的小鼠丢失了大约1kb的序列外,其余品系小鼠的重组产物与预想的结构符合.在当代和后代的转基因小鼠乳汁中均可测到氯霉素乙酰化酶的活性.这表明融合基因在转基因小鼠乳腺中得到表达和分泌,也说明显微共注射两个相互重叠的基因片段是建立转基因动物的一个可行途径.     

转基因动物技术及其若干问题

转基因动物是用基因工程技术,将外源目的基因经由生殖细胞或早期胚胎导入动物胚胎染色体基因组内,使之稳定整合并能传给后一代的动物。被导入的外源目的基因称为转基因。经转基因操作成功的受精卵或胚胎所发育的第一代个体称为首建者。1974年Jaenisch等首次报道应用显微注射法将SV40注入鼠胚胎获得第一例嵌合型转基因小鼠。至今,转基因动物     

精子载体法制备转基因动物的研究进展

精子载体法是一种低成本、简单快速制备转基因动物的方法.外源基因主要通过两种方式实现基因整合,一种是直接整合到精子的基因组中;另一种是通过精子携带进入卵细胞,然后整合到受精卵的基因组中.尽管现在已经有很多利用精子载体法获得了转基因动物的研究,但是基于精子在受精过程中,结合、内化、以及转运外源基因等方面能力的差异,转染效率仍有待提高.就利用精子载体法制备转基因动物过程中,精子载体法制备转基因动物的分子机制和方法方面进行系统的阐述和分析.     

精子因素对精子载体法制备转基因山羊的影响

精子具有主动结合、转运、整合外源DNA的能力,并在受精时导入卵母细胞,获得转基因动物.精子介导基因转移(sperm-mediated gene transfer,SMGT)是目前获得转基因动物简单而高效的方法之一.精子因素是影响SMGT方法生产转基因动物的重要方面.本论文结合我们的研究针对转染用山羊(Capra hircus)精液的来源、精子质膜完整性、精液品质及发育阶段等精子因素影响精子结合外源DNA和SMGT方法生产转基因山羊的效率进行了论述,并从这些影响因素入手,提出了筛选精子供体、保持精液品质、调控质膜等措施,提高精子转染外源DNA能力和生产转基因动物的效率.     

水稻胚与胚乳分化发育中的内源多胺

稻胚发育过程中,其内源多胺以腐胺、亚精胺为主。在幼胚分化期,腐胺和亚精胺的含量很高;幼胚分化完成时,其含量急剧下降;直至分化后期才趋稳定。在胚及胚乳发育时期,还出现一种未知多胺X_(22),其含量除在胚分化完成时较少外,在胚发育的其他各期中,含量则一直很高。DNA和蛋白质含量的变化,从分化期开始递增直至物质积累成熟期,其趋势均相同。多胺可能参与胚与胚乳中核酸和蛋白质合成的调节。     

Morphology of the regiones ethmoidalis and orbitotemporalis in Galea musteloides Meyen 1832 and Kerodon rupestris (Wied-Neuwied 1820) (Rodentia: Caviidae) with comments on the phylogenetic systematics of the Caviidae

The present study describes the fetal regions ethmoidalis and orbitotemporalis of the cranium of Galea musteloides and Kerodon rupestris and compares them with Galea spixii and Cavia porcellus . The fetal crania of these representatives of the Caviinae were serially sectioned and 3D plate reconstructions were built. For a broader scope, serially sectioned heads of the caviomorph taxa Hydrochaeris hydrochaeris and Dasyprocta leporina were also examined. The ethmoidal and orbitotemporal regions of the cranium provides characters that are pertinent to the discussion of systematics of the Caviinae. Nine cranial characters were identified as being significant and discussed phylogenetically. Taking the monophyly of the Cavioidea as given, two characters are autapomorphic for the genus Galea: the reduction of the crista semicircularis and the lacrimale forming the 'arco antorbitario' (Kraglievich 1930). Cavia is the sister group of Galea; synapomorphic characters are the extent of the processus palatinus medialis of the praemaxillare and the presence lamina pterygoidea of the alisphenoid. There were no apomorphic characters of the structural complexes examined that could support the Caviinae. The reduced and nonperforated alisphenoid is an autapomorphic feature of the Caviidae. The division of the nasoturbinale is evidence for a sistergroup relationship of the Hydrochaeridae and the Caviidae. Autapomorphic characters for the Cavioidea include the ventrolateral opening of the organon vomeronasale and the lateral prolongation of the lacrimale.     

Biocontrol of Pests of Apples and Pears in Northern and Central Europe: 1. Microbial Agents and Nematodes

The viral, bacterial, fungal and nematode pathogens of arthropod pests of apple and pear in northern and central Europe and their use as biocontrol agents are reviewed. Baculoviruses are important viral pathogens of several lepidopterous pests of apple and pear but other viral pathogens have not been investigated in depth and are little known. The granuloviruses of codling moth, Cydia pomonella (CpGV), and to a lesser extent, of the summer fruit tortrix moth, Adoxophyes orana (AoGV), have been researched extensively and are exploited as biological control agents. Commercial development and use has been limited because of their high costs, slow action, short persistence and specificity relative to broad-spectrum pesticides. The widespread development of strains of codling moth multi-resistant to insecticides and the desire to reduce dependence on pesticides have improved the commercial prospects of CpGV and use is likely to increase. The development of a genetically improved egt-strain of CpGV (lacking the ecdysteroid-UDP glucosyl transferase gene) in the UK is a significant breakthrough, though commercialization in the UK may be difficult due to adverse public attitudes to the release of genetically-modified microorganisms. Future research and development approaches include further genetic manipulation of CpGV and AoGV to improve potency, speed of kill and/or persistence, improvement of formulation (to reduce UV light sensitivity) and development of cheaper mass production techniques and possibly in vitro production. A systematic search for baculoviruses and other viruses of apple and pear pests is likely to reveal important new opportunities. The most important bacterial pathogen used as a biological control agent is Bacillus thuringiensis (Bt). However, Bt products currently available have limited effectiveness against many orchard pests due to the pests' cryptic life habits. The HD-1 Bt strain has been investigated and used extensively for control of leaf-rolling tortricid larvae and is widely used, but efficacy is moderate. Advances in biotechnology and genetic engineering provide opportunity for development of Bt strains designed specifically to control orchard pests, but this has not yet been done for commercial reasons. Other research approaches include the evaluation of new Bt products developed for other markets worldwide and the bioassay of strains from Bt collections against specific apple or pear pests. Entomopathogenic fungi provide good opportunity for development as biological control agents of apple and pear pests. The main factor limiting their effectiveness is the requirement for high humidities and moderate temperatures for spore germination and development. For foliar pests, a useful starting point for research might be the control of sucking pests which excrete honeydew (e.g. Cacopsylla sp. or aphids) or those that inhabit protected microenvironments (e.g. Dasineura sp.). Key areas for research are improved formulation, the selection of low temperature-active strains, field evaluation and avoiding possible adverse effects of fungicides. An alternative approach is to examine the exploitation of entomopathogenic fungi in soil, to which many species of entomopathogenic fungi are adapted ecologically. Apple and pear orchards provide long-term stable habitats where populations of entomopathogenic fungi in soil are likely to be large. There are few important soil pests of apple or pear. However, many species spend part of their life in soil, mainly to pupate or overwinter, where they may be targeted by fungal entomopathogenic biocontrol agents. Entomopathogenic nematodes have many attributes which favour them as biological control agents. However, their requirement for surface moisture for survival and movement means there are only limited prospects for using them as biological control agents for foliar pests. As with entomopathogenic fungi, there are better prospects for control of pests that occur in soil. Microbial pathogens and entomopathogenic nematodes are important components of the natural enemy complex of apple and pear orchards and more effort needs to be devoted to fostering them and exploiting them as biocontrol agents in sustainable, biologically-based Integrated Pest Management programmes. They can in many cases be mass produced at low cost by bulk fermentation processes and applied as sprays (as 'biopesticides') and are, at least potentially, ideal biological control agents for many apple or pear pests. Important general characteristics are their comparative environmental and human safety, compatibility with other control strategies in Integrated Pest Management programmes and reproductive capacity. They tend to be effective in a narrower range of environmental conditions than pesticides, but there is considerable potential to improve their effectiveness by improved formulation, strain selection and genetic manipulation. They are often host-specific and thus, offer restricted marketing opportunities, which is a significant barrier to development and commercialisation. Registration procedures and associated fees for microbial agents are a further significant barrier. Such requirements do not apply currently to nematodes.     

Areography of the Gymnosperms of China (1) Distribution of the Pinaceae of China

Nine of 10 genera and 119 of approximately 240 species of the Pinaceae occur in China, including 67 endemic species and two endemic genera. In this paper, the distributional maps of all the genera of the Pinaceae are presented (fig. 1-8). The horizontal and vertical distributions of species in each genus are discussed. The analysis of the distribution patterns of the genera indicates that some genera, such as Keteleeria, Tsuga, Pseudotsuga, Cathaya and Pseudolarix, are restricted to the area south of the Qinling Mountains and the Huaihe River, and the others, i. e. Picea, Abies, Larix and Pinus, extend northward to northeastern China. However, all of the genera except Keteleeria and Pinus are not found in very dry areas and tropical mountainous regions of China. The monotypic genera, Cathaya and Pseudolarix, are distributed in eastern and central China. The genus Keteleeria consists of 10 species, 7 of which are concentrated in southern Guizhou, northern Guangxi, southwestern Hunan and easternmost Yunnan. The distribution of the remaining 6 genera shows the maximum concentration in western Sichuan and northwestern Yunnan. (Figs. 2-8). Furthermore, more than third of species of the Pinaceae (37.8%) are also concentrated in western Sichuan and northwestern Yunnan. where a great variety of habitats and different topographic features occur. It is apparent that to conduct our systematic and evolutionary studies on this family in these region is especially needed. The relations between the areal size and the tolerance of species are discussed. The distributions of macrofossils and microfossils of the genera of the Pinaceae ia China are given, and it has been proved that areas of most genera of the family were considerably larger in the past. than at present.     

中国刺猬的染色体研究

本文采用全血培养和骨髓染色体制片法,对分布于我国南京市郊和济南市郊的刺猬染色体进行了组型、C-带、G-带和银染色的观察分析,并与东欧、西欧两种刺猬比较,它们之间的核型及带型差异显著;又将南京、济南及金清波(1985)报道的河南新乡三地分布的刺猬进行比较,它们的核型及带型也显示出一定差异,这种多态性在分类和进化上有一定的意义。     

Cdc42 protein acts upstream of IQGAP1 and regulates cytokinesis in mouse oocytes and embryos

Cdc42 and Rac1 Rho family GTPases, and their interacting protein IQGAP1 are the key regulators of cell polarity. We examined the role of Cdc42 and IQGAP1 in establishing the polarity of mouse oocyte and regulation of meiotic and mitotic divisions. We showed that Cdc42 was localized on the microtubules of meiotic and mitotic spindle and in the cortex of mouse oocytes and cleaving embryos. IQGAP1 was present in the cytoplasm and cortex of growing and fully-grown oocytes. During maturation it disappeared from the cortex and during meiotic and mitotic cytokinesis it concentrated in the contractile ring. Toxin B inhibition of the binding activity of Cdc42 changed the localization of IQGAP1, inhibited emission of the first polar body, and caused disappearance of the cortical actin without affecting the migration of meiotic spindle. This indicates, that in maturing oocytes accumulation of cortical actin is not indispensable for spindle migration. In zygotes treated with toxin B actin cytoskeleton was rearranged and the first and/or subsequent cytokinesis were inhibited. Our results indicate that Cdc42 acts upstream of IQGAP1 and is involved in regulation of cytokinesis in mouse oocytes and cleaving embryos, rather than in establishing the polarity of the oocyte.     

Carboxylesterases in lipid metabolism: from mouse to human

Mammalian carboxylesterases hydrolyze a wide range of xenobiotic and endogenous compounds, including lipid esters. Physiological functions of carboxylesterases in lipid metabolism and energy homeostasis in vivo have been demonstrated by genetic manipulations and chemical inhibition in mice, and in vitro through (over)expression, knockdown of expression, and chemical inhibition in a variety of cells. Recent research advances have revealed the relevance of carboxylesterases to metabolic diseases such as obesity and fatty liver disease, suggesting these enzymes might be potential targets for treatment of metabolic disorders. In order to translate pre-clinical studies in cellular and mouse models to humans, differences and similarities of carboxylesterases between mice and human need to be elucidated. This review presents and discusses the research progress in structure and function of mouse and human carboxylesterases, and the role of these enzymes in lipid metabolism and metabolic disorders.     

Taxonomic, evolutionary and ecological implications of the leaf anatomy of rhizomatous Iris species

The leaf anatomy of the rhizomatous Iris species with ensiform leaves and the related genera Pardanthopsis and Belamcanda is described. Their isobilateral leaves may or may not have a pseudo-dorsiventral structure. Variable characters of their leaf blades include: outline in transverse section, height and shape of papillae, form and structure of stomata, transverse section outline of marginal fibre strands and sclerenchy matous inner bundle sheath at phloem and xylem poles, forms of mesophyll arrangement, mesophyll structure and air canals, vascular bundle arrangement and the detailed structure of the larger vascular bundles, distribution of tannin, size and distribution of crystals. The taxonomic and ecological significance of these characters has been evaluated.
The anatomical characteristics of 25 supraspecific taxa in three genera are presented and compared in tables. The relationships and evolutionary position of these taxa are discussed. Each of the three subgroups within Iris appears to be correlated with a syndrome of anatomical characters. Some species currently of uncertain taxonomic position are discussed, and their classification based on anatomical data is suggested.
Some characters related to xeromorphy or helomorphy are mentioned.     

几种党参属植物及其近缘种花粉形态的比较

对6种桔梗科（党参属Codonopsis4个种以及桔梗属Platycodon和风铃草属Campanula各1种）花粉进行了光镜及扫描电镜的观察比较,发现党参属不同种花粉的形态特征很不相同,易于区别,如党参花粉极面观多为7裂圆形,外壁形面具乳突状的短刺,川党参花粉极面观圆形,刺在沟的两旁排成两列,小花党参花粉极面观近圆形至7裂圆形,外壁表面的刺比其它种的都大,鸡蛋参花粉外壁表面的刺柔软而稍为弯曲,其长度为上述各种刺的数倍,桔梗属和风铃草属花粉与党参属花粉的差异更大,桔梗属花粉粒扁球形,极面观五角形,少数四或六角形,风铃草属花粉粒更为特殊,极面观近圆三角形,具3孔,花粉外壁的刺不仅锐利而且很粗,花粉形态在党参属不同种间以及党参属与桔梗属和风铃草属之间的差异为其分类学提供了进一步的孢粉学资料。