家兔个体表达系统的建立

将乙肝表面抗原基因及人生长激素基因用微注射法转入家兔受精卵发育成为有整合,有表达的当代兔,并繁殖出了有外源基因存在,有表达的子代兔,转基因所用的质粒是含（1）乙肝病毒表面抗S基因与小鼠MT启动子的pMTSA.(2)乙肝病毒核心抗原基因和乙肝病毒S基因启动子的pHBV3.0,和（3）含有人生长激素基因SV40启动子和小鼠MT启动子的pSMGH,质粒均切成线性人子后再注射,从757个微注射并移植的受精卵经发良娩出得101仔兔。其中有57％具有整合的微注射基因,用ELISA法测出28只转基因兔内有8只的血清中有表达的乙肝病毒表面抗原,占总数约30％,转基因兔与转基因兔和其与正常兔交配获得的子代中有83％含有转基因,有15％在血清中可测出乙型肝炎表面抗原,对转入人生长激素基因的兔也作了整合和表达的检测。     

蓝斑区去甲肾上腺素能神经元在Orexin 促麻醉觉醒中的作用研究

目的：探讨蓝斑区(LC)去甲肾上腺素能神经元在orexin 促麻醉觉醒中作用。方法：应用异氟烷对成年SD 大鼠进行麻醉,15 分钟后,将SD 大鼠随机分为6 组,分别注射orexin-A/B (100pmol/0.3 滋L) 及其溶剂saline (0.3 滋L);orexin I 型受体拮抗剂 SB334867/ II型受体拮抗剂TCS-OX2-29(20 滋g/0.3 滋L及其溶剂DMSO(0.3 滋L),通过观察大鼠翻正反射的消失和恢复时间,研究 蓝斑区微注射orexin 及其拮抗剂对异氟烷麻醉的诱导和觉醒的影响。结果：蓝斑区(LC)微注射四种试剂或其溶剂均对SD 大鼠异 氟烷麻醉的诱导时间无明显影响;蓝斑区(LC)微注射orexin-A 能缩短SD 大鼠异氟烷麻醉觉醒时间(P<0.001),而微注射orexinI 型拮抗剂SB334867 能延长觉醒时间(P<0.001);orexin-B、orexin II型受体拮抗剂TCS-OX2-29 对大鼠异氟烷麻醉的觉醒无明显影 响。结论：蓝斑区(LC)的去甲肾上腺素能神经元介导了orexin 的促麻醉觉醒作用。     

Cleavage development of bovine oocytes fertilized by sperm injection

Whole in vitro capacitated bovine spermatozoa were microinjected directly into the ooplasm of in vitro matured bovine oocytes in order to determine whether oocytes fertilized by sperm injection could undergo normal pronuclear formation and cleavage development. Immature oocytes recovered from follicles (2-5 mm) of unstimulated ovaries were cultured for 24-25 h in modified TCM 199 medium supplemented with heat-treated day 20 cow serum, luteinizing hormone (LH), and estradiol 17-B. In vitro capacitated, frozen-thawed spermatozoa were injected into the ooplasm, and the injected oocytes were cultured for an additional 24-28 h. Twenty-one percent (21/101) of the sperm-injected oocytes contained a sperm within the ooplasm; however, only 2% (2/101) cleaved. The remaining oocytes either did not contain a sperm or had degenerated. After oocyte activation induced by a 5 min incubation in 1 microM A23187, sperm nuclear decondensation occurred in the A23187-activated, injected oocytes but not in the unactivated, injected controls (37% vs. 0% after 3 h). Those injected, activated oocytes that contained a male pronucleus also exhibited a female pronucleus and second polar body. Furthermore, a significantly higher number (28%, 6/21) of the injected, activated oocytes cleaved to a two- to four-cell stage after 48 h than did the injected, unactivated oocytes (4%). These results indicate that, unlike hamster and rabbit oocytes, bovine oocytes are not sufficiently stimulated by the injection procedure to complete meiosis, but, upon activation by calcium ionophore, they will undergo normal-appearing cleavage development following fertilization by sperm injection.     

Identification of embryogenic microspores of barley (Hordeum vulgare L.) by individual selection and culture and their potential for transformation by microinjection

Summary In order to identify microspores, suitable for transformation via microinjection of DNA, single microspores of barley (Hordeum vulgare L.) were selected after initial preculture of anthers floating on liquid media and analysed for their development in individual culture in microdroplets of culture medium. Conditions for microculture and plant regeneration from single selected embryogenie microspores were established. The technical feasability of intranuclear microinjection was demonstrated by injecting the fluorescent dye Lucifer Yellow. All essential procedures for a transformation system of barley based on microinjection into microspores have thus been performed successfully. Further efforts to increase efficiencies of culture and microinjection procedures are necessary, however, in order to improve the suitability of this approach towards transformation of barley.Abbreviations MES 2 (N-morpholino) ethanesulfonic acid - PEG polyethylene glycol     

转基因家兔模型制作方法

作为生物医学研究重要的实验动物模型,转基因家兔已经被广泛应用在人类心脑血管疾病、艾滋病以及癌症等生物医学研究领域,特别是利用转基因家兔模型在人类动脉粥样硬化实验研究中已经取得了令人注目的成绩。本文结合我们自己制作转基因家兔的经验、研究成果以及文献资料,详细介绍了利用原核显微注射法、直接将外源基因注入受精卵雄原核中的转基因家兔制作技术,回顾了利用转基因家兔模型在生物医学研究中取得的重要进展。     

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die¹. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm². The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death^3-4.The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria⁵. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells^6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis^10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.     

Actins from plant and animal sources tend not to form heteropolymers in vitro and function differently in plant cells

Summary. Pollen and skeletal muscle actins were purified and labeled with fluorescent dyes that have different emission wavelengths. Observation by electron microscopy shows that the fluorescent actins are capable to polymerize into filamentous actin in vitro, bind to myosin S-1 fragments, and have a critical concentration similar to unlabeled actin, indicating that they are functionally active. The globular actins from two sources were mixed and polymerized by the addition of ATP and salts. The copolymerization experiment shows that when excited by light of the appropriate wavelength, both red actin filaments (pollen actin) and green actin filaments (muscle actin) can be visualized under the microscope, but no filaments exhibiting both green and red colors are detected. Furthermore, coprecipitations of labeled pollen actin with unlabeled pollen and skeletal muscle actin were performed. Measurements of fluorescent intensity show that the amount of labeled pollen actin precipitating with pollen actin was much higher than that with skeletal muscle actin, indicating that pollen and muscle actin tend not to form heteropolymers. Injection of labeled pollen actin into living stamen hair cells results in the formation of normal actin filaments in transvacuolar strands and the cortical cytoplasm. In contrast, labeled skeletal muscle actin has detrimental effects on the cellular architecture. The results from coinjection of the actin-disrupting reagent cytochalasin D with pollen actin show that overexpression of pollen actin prolongs the displacement of the nucleus and facilitates the recovery of the nuclear position, actin filament architecture, and transvacuolar strands. However, muscle actin perturbs actin filaments when injected into stamen hair cells. Moreover, nuclear displacement occurs more rapidly when cytochalasin D and muscle actin are coinjected into the cell. It is concluded that actins from plant and animal sources behave differently in vitro and in vivo and that they are functionally not interchangeable.     

DNA-induced endocytosis upon local microinjection to giant unilamellar cationic vesicles

We suggest a novel approach for direct optical microscopy observation of DNA interaction with the bilayers of giant cationic liposomes. Giant unilamellar vesicles, about 100 μm in diameter, made of phosphatidylcholines and up to 33 mol% of the natural bioactive cationic amphiphile sphingosine, were obtained by electroformation. “Short” DNAs (oligonucleotide 21b and calf thymus 250 bp) were locally injected by micropipette to a part of the giant unilamellar vesicle (GUV) membrane. DNAs were injected native, as well as marked with a fluorescent dye. The resulting membrane topology transformations were monitored in phase contrast, while DNA distribution was followed in fluorescence. We observed DNA-induced endocytosis due to the DNA/lipid membrane local interactions and complex formation. A characteristic minimum concentration (C _endo) of d-erythro-sphingosine (Sph⁺) in the GUV membrane was necessary for the endocytic phenomenon to occur. Below C _endo, only lateral adhesions between neighboring vesicles were observed upon DNA local addition. C _endo depends on the type of zwitterionic (phosphocholine) lipid used, being about 10 mol% for DPhPC/Sph⁺ GUVs and about 20 mol% for SOPC/Sph⁺ or eggPC/Sph⁺ GUVs. The characteristic sizes and shapes of the resulting endosomes depend on the kind of DNA, and initial GUV membrane tension. When the fluorescent DNA marker dye was injected after the DNA/lipid local interaction and complex formation, no fluorescence was detected. This observation could be explained if one assumes that the DNA is protected by lipids in the DNA/lipid complex, thereby inaccessible for the dye molecules. We suggest a possible mechanism for DNA/lipid membrane interaction involving DNA encapsulation within an inverted micelle included in the lipid membrane. Our model observations could help in understanding events associated with the interaction of DNA with biological membranes, as well as cationic liposomes/DNA complex formation in gene transfer processes. Received: 18 April 1998 / Revised version: 6 August 1998 / Accepted: 7 August 1998