In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease and injury regional estimates, World Health Organization, 2004). Surgeries that often compromise the quality of life are required to correct heart defects, reminding us of the importance of finding the causes of CHD. Mutant mouse models and live imaging technology have become essential tools to study the etiology of this disease. Although advanced methods allow live imaging of abnormal hearts in embryos, the physiological and hemodynamic states of the latter are often compromised due to surgical and/or lengthy procedures. Noninvasive ultrasound imaging, however, can be used without surgically exposing the embryos, thereby maintaining their physiology. Herein, we use simple M-mode ultrasound to assess heart rates of embryos at E18.5 in utero. The detection of abnormal heart rates is indeed a good indicator of dysfunction of the heart and thus constitutes a first step in the identification of developmental defects that may lead to heart failure.     

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at ≥6 months after birth. We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.     

抗EHFV的独特型抗体在小鼠体内诱导免疫应答的研究

本文介绍用流行性出血热（EHF）病毒J10株制备单克隆抗体（McAb）,以及用7个McAb免疫家兔,使其产生抗EHF病毒McAb的抗体即抗独特型 抗体（Ab2).Ab2能在体外同出血热病人恢复期血清和EHF病毒免疫的兔血清发生特异性结合。再经EHF－McAb亲和层析法分离提纯Ab2,免疫BALb/c小鼠,将所获得的免疫血清（Ab3）用荧光和ELISA分别加以测定。结果表明,抗－抗独特型抗体可在体外识别EHF病毒,而不能同病病人恢复期血清发生结合,从而支持免疫网络学说,可能为我们提供一种新型的抗原来源途径。     

Role of the Melanocyte in Epidermal Inflammatory and Immune Responses

The epidermis is composed of four major cell types in the mouse. In addition to keratinocytes and Langerhans cells, there are also melanocytes and Thyl⁺ lymphocytes. We propose that for the epidermis to maintain homeostasis, all four cell types must interact together in an integrated fashion. Vitiligo is a form of depigmentation that affects human subjects. Depigmentation is common in the animal kingdom. In at least several animal species, depigmentation is accompanied by loss of responsiveness of the epidermis to potent contact allergens like dinitrofluorobenzene. The C57Bl/Ler vit/vit mouse spontaneously depigments and is phenotypically similar to humans with vitiligo. We have studied this species extensively and have found that it has an isolated immune defect to contact allergens. In this report, we document that there is loss of epidermal melanocytes. Although there is a loss of epidermal contact sensitivity in this animal, other immune parameters such as dermal delayed-type hypersensitivity are normal. We attribute this loss of immune sensitivity in the epidermis to be associated with loss of the melanocyte. We report and review in detail the many peptides and other types of inflammatory mediators that affect both melanocyte function as well as immune responsiveness.     

The Role of Intracellular pH in Fertilization of Sand Dollar Eggs Analyzed by Microinjection Method

The change in intracellular pH (pH_i) upon fertilization and the effects of changing the pH_i by microinjection of pH buffers were investigated in the eggs of the sand dollar, Clypeaster japonicus. The pH_i was determined by the tint of a pH indicator, phenol red, microinjected into eggs. The pH_i ranged from 6.5 to 6.75 in unfertilized eggs and it rose by 0.4 to 0.5 unit within 3 min upon fertilization. The elevated pH_i ranging from 7.0 to 7.25 was maintained at least until the first cleavage. As reported in eggs of other species of sea urchin (1–4), development of fertilized eggs which had been transferred to Na-free sea water immediately after insemination was arrested and the pH_i did not rise remaining at the level of unfertilized eggs. Development was initiated in eggs arrested in Na-free sea water when the pH_i was elevated up to the level of fertilized eggs, i.e. 7.0 to 7.25, by microinjecting 1 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-KOH buffer at pH 8.0. By microinjection of pH 7.5 buffer, some eggs started development though none of them underwent cleavage. By microinjection of pH 7.0 or pH 6.5 buffer, development was not initiated. The initiation of development depended on the pH value of microinjected pH buffer, and in consequence, on the final pH_i. The elongation of microvilli which had been arrested in eggs in Na-free sea water was also induced by microinjection of pH 8.0 or 7.5 buffer.     

雷帕霉素靶在小鼠受精卵早期发育中的作用研究

哺乳动物雷帕霉素靶(mTOR)是细胞生长的中心调控因子,应用RT-PCR、免疫印迹、放射性同位素体外测定酶活性等方法,研究mTOR在小鼠受精卵第一次有丝分裂过程中在卵中的表达、活性变化以及对卵裂的影响.研究发现mTOR在小鼠卵母细胞和受精卵中都有表达,在mRNA水平,mTOR从G2期开始降解,在蛋白水平,则各期没有明显变化;mTOR的激酶活性在受精后明显升高,并且在整个1-细胞期保持较高活性;mTOR的特异性抑制剂雷帕霉素能抑制卵裂,并且能抑制成熟促进因子MPF的调节亚基cyclin B的表达,从而抑制了MPF的活性.结果表明mTOR可能通过促进MPF的激活而促进小鼠受精卵的分裂.     

In Vivo Role of Phosphorylation of Cryptochrome 2 in the Mouse Circadian Clock

The circadian clock is finely regulated by posttranslational modifications of clock components. Mouse CRY2, a critical player in the mammalian clock, is phosphorylated at Ser557 for proteasome-mediated degradation, but its in vivo role in circadian organization was not revealed. Here, we generated CRY2(S557A) mutant mice, in which Ser557 phosphorylation is specifically abolished. The mutation lengthened free-running periods of the behavioral rhythms and PER2::LUC bioluminescence rhythms of cultured liver. In livers from mutant mice, the nuclear CRY2 level was elevated, with enhanced PER2 nuclear occupancy and suppression of E-box-regulated genes. Thus, Ser557 phosphorylation-dependent regulation of CRY2 is essential for proper clock oscillation in vivo.     

In vivo inhibition of nitrogenase by hydroxylamine in Rhodospirillaceae Role of nitric oxide

Rhodobacter capsulatus strains E1F1 and B10 and Rhodobacter sphaeroides DSM 158 did not use hydroxylamine as nitrogen source for growth but metabolized it mainly through the glutamine synthetase reaction. Hydroxylamine had a high toxicity for cells growing either under phototrophic or dark-aerobic conditions. l-methionine-d,l-sulfoximine partially inhibited hydroxylamine uptake and increased the inhibition time of nitrogenase activity by this nitrogen compound. Nitric oxide was also a powerful inhibitor of nitrogenase in intact cells of R. capsulatus. Since low amounts of NO were produced from hydroxylamine, short-term inhibition of nitrogenase in the presence of this compound could be mediated in vivo by nitric oxide.Abbreviations GS glutamine synthetase - MSX l-methionine-d,l-sulfoximine - MTA mixed alkyltrimethylammonium bromide     

In utero characterisation of fetal growth by ultrasound scanning in the rabbit

Fetal development is an important factor influencing the susceptibility of adults to metabolic diseases. In order to study the influence of fetal growth on further development in animal models like the rabbit, methods of measurement of fetal and placental size and viability must be established and validated. In this study, 42 New Zealand does bred naturally (N=12) or transferred with in vivo produced embryos (2, 4 or 6 embryos/doe) have been scanned every 2-3 days with a 7.5 MHz transabdominal probe from Day 7 post-coitum until term to measure fetal and placental growth. Vesicle, placental, fetal length and head size have thus been determined according to number of fetuses and time. In late gestation, the fetuses that were transferred in limited numbers to the uterus of does were significantly larger than their natural breeding counterparts probably due to reduced litter size.     

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system ^1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex ^6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method ⁹. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice ^10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA ¹². These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.     

Development of a Mouse Monoclonal Antibody Cocktail for Post-exposure Rabies Prophylaxis in Humans

As the demand for rabies post-exposure prophylaxis (PEP) treatments has increased exponentially in recent years, the limited supply of human and equine rabies immunoglobulin (HRIG and ERIG) has failed to provide the required passive immune component in PEP in countries where canine rabies is endemic. Replacement of HRIG and ERIG with a potentially cheaper and efficacious alternative biological for treatment of rabies in humans, therefore, remains a high priority. In this study, we set out to assess a mouse monoclonal antibody (MoMAb) cocktail with the ultimate goal to develop a product at the lowest possible cost that can be used in developing countries as a replacement for RIG in PEP. Five MoMAbs, E559.9.14, 1112-1, 62-71-3, M727-5-1, and M777-16-3, were selected from available panels based on stringent criteria, such as biological activity, neutralizing potency, binding specificity, spectrum of neutralization of lyssaviruses, and history of each hybridoma. Four of these MoMAbs recognize epitopes in antigenic site II and one recognizes an epitope in antigenic site III on the rabies virus (RABV) glycoprotein, as determined by nucleotide sequence analysis of the glycoprotein gene of unique MoMAb neutralization-escape mutants. The MoMAbs were produced under Good Laboratory Practice (GLP) conditions. Unique combinations (cocktails) were prepared, using different concentrations of the MoMAbs that were capable of targeting non-overlapping epitopes of antigenic sites II and III. Blind in vitro efficacy studies showed the MoMab cocktails neutralized a broad spectrum of lyssaviruses except for lyssaviruses belonging to phylogroups II and III. In vivo, MoMAb cocktails resulted in protection as a component of PEP that was comparable to HRIG. In conclusion, all three novel combinations of MoMAbs were shown to have equal efficacy to HRIG and therefore could be considered a potentially less expensive alternative biological agent for use in PEP and prevention of rabies in humans.     

Role of c-kit in mouse spermatogenesis: identification of spermatogonia as a specific site of c-kit expression and function.

Recent studies have shown that the dominant white spotting (W) locus encodes the proto-oncogene c-kit, a member of the tyrosine kinase receptor family. One symptom of mice bearing mutation within this gene is sterility due to developmental failure of the primordial germ cells during early embryogenesis. To elucidate the role of the c-kit in gametogenesis, we used an anti-c-kit monoclonal antibody, ACK2, as an antagonistic blocker for c-kit function to interfere with the development of male and female germ cells during postnatal life. ACK2 enabled us to detect the expression of c-kit in the gonadal tissue and also to determine the functional status of c-kit, which is expressed on the surface of a particular cell lineage. Consistent with our immunohistochemical findings, the intravenous injection of ACK2 into adult mice caused a depletion in the differentiating type A spermatogonia from the testis during 24-36 h, while the undifferentiated type A spermatogonia were basically unaffected. Intraperitoneal injections of ACK2 into prepuberal mice could completely block the mitosis of mature (differentiating) type A spermatogonia, but not the mitosis of the gonocytes and primitive type A spermatogonia, or the meiosis of spermatocytes. Our results indicate that the survival and/or proliferation of the differentiating type A spermatogonia requires c-kit, but the primitive (undifferentiated) type A spermatogonia or spermatogenic stem cells are independent from c-kit. Moreover, the antibody administration had no significant effect on oocyte maturation despite its intense expression of c-kit.