Importing, caring, breeding, genotyping, and phenotyping a genetic mouse in a Chinese university.

The genetic manipulation of the laboratory mouse has been well developed and generated more and more mouse lines for biomedical research. To advance our science exploration, it is necessary to share genetically modified mouse lines with collaborators between institutions, even in different countries. The transfer process is complicated. Significant paperwork and coordination are required, concerning animal welfare, intellectual property rights, colony health status, and biohazard. Here, we provide a practical example of importing a transgenic mice line, Dynamin 1 knockout mice, from Yale University in the USA to Perking University in China for studying cell secretion. This example including the length of time that required for paper work, mice quarantine at the receiving institution, and expansion of the mouse line for experiments. The procedure described in this paper for delivery live transgenic mice from USA to China may serve a simple reference for transferring mouse lines between other countries too.

Effect of cocaine on ion channels and glutamatergic EPSCs in noradrenergic locus coeruleus neurons.

The locus coeruleus (LC) is an important brainstem area involved in cocaine addiction. However, evidence to elucidate how cocaine modulates the activity of LC neurons remains incomplete. Here, we performed whole recordings in brain slices to evaluate the effects of cocaine on the sodium (Na(+)), potassium (K(+)), calcium (Ca(2+)) channels, and glutamatergic synaptic transmission in the locus coeruleus neurons. Local application of cocaine significantly and reversibly reduced the spontaneous firing rate but did not affect action potential amplitude, rising time, decay time, or half width of noradrenergic locus coeruleus neurons. Moreover, cocaine attenuated the sodium current but did not affect potassium and calcium currents. The N-methyl-D-aspartate receptor mediated excitatory postsynaptic currents were reduced by neuropeptide galanin but not cocaine. All those data demonstrate that cocaine has inhibitory effect on the spontaneous activities and sodium current in locus coeruleus neurons. Therefore, neuromodulation of sodium channel in locus coeruleus neurons may play an important role in drug addiction.

The development of a loop-mediated isothermal amplification assay for rapid and sensitive detection of abalone herpesvirus DNA.

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of abalone herpesvirus DNA. Two pairs of primers were designed, based on the sequence of the DNA polymerase gene of abalone herpesvirus. The reaction temperature and time were optimized to 63°C and 60min, respectively. LAMP amplicons were analyzed by 2% agarose gel electrophoresis or by visual inspection of a colour change emitted by fluorescent dye. The method developed was specific for the detection of abalone herpesvirus, without cross-reactions with other tested herpesviruses including ostreid herpesvirus 1 (OsHV-1), European eel herpesvirus, koi herpesvirus (KHV) and an avian herpesvirus. The LAMP assay was 100 folds more sensitive than a conventional PCR and 10 folds less sensitive than a SYBR Green PCR. These results indicate that the developed LAMP assay is a simple, rapid, sensitive, specific and reliable technique for the detection of abalone herpesvirus.

Development of a polymerase chain reaction for the detection of abalone herpesvirus infection based on the DNA polymerase gene.

A 5781-base pair (bp) fragment of genomic DNA from the Taiwanese abalone herpesvirus was obtained and showed 99% (5767/5779) homology in the nucleotide sequence and 99% (1923/1926) in the amino acid sequence with the DNA polymerase gene of the abalone herpesvirus strain Victoria/AUS/2007. Homology of the amino acid sequence with the DNA polymerase of ostreid herpesvirus 1 was 30% (563/1856). In this study, a PCR-based procedure for detecting herpesvirus infection of abalone, Haliotis diversicolor supertexta, in Taiwan was developed. The method employed primer sets targeting the viral DNA polymerase gene, and was able to amplify DNA fragments of the expected size from infected samples. Primer sets of 40f and 146r were designed for amplification of an expected PCR product of 606 bp. Combining the new PCR protocol with histopathology, this assay can serve as a reliable diagnostic for herpesvirus infections in abalone.

Differential effects of propofol and ethanol on P2X4 receptors expressed in Xenopus oocytes.

The current study investigated the effects of propofol on P2X4 receptors expressed in Xenopus oocytes using two-electrode voltage clamp. We also tested the effects of 100 mM ethanol on the same oocytes used to test propofol. Propofol potentiated ATP-gated currents in a concentration dependent manner in P2X4 receptors. In agreement with our previous findings, ethanol inhibited P2X4 receptors. The opposite effects of propofol and ethanol on P2X4 receptor function suggest that these anesthetics act via different sites/mechanisms in P2X receptors as has been suggested for GABA(A) and glycine receptors.

TSH and TSH receptor antibody-binding sites in fibroblasts of pretibial myxedema are related to the extracellular domain of entire TSH receptor.

The role of TSH receptor antibodies in the pathogenesis of pretibial myxedema is still unclear. This study was designed to determine whether patients with pretibial myxedema had higher serum titers of TSH receptor antibodies, and whether there were TSH and TSH receptor antibody-binding sites on plasma membranes of fibroblasts derived from the skin of pretibial myxedema. If there were, were the binding sites similar to the TSH receptor? The TSH receptor antibodies were determined with radioreceptor assay in 20 normal subjects, 18 hyperthyroid Graves' disease patients without ophthalmopathy, 26 hyperthyroid Graves' disease patients with ophthalmopathy, and 11 patients with pretibial myxedema associated with Graves' ophthalmopathy. TSH and TSH receptor antibody-binding sites were studied on plasma membranes of fibroblasts cultured from the skin of pretibial myxedema with radioreceptor assay. RNA was also extracted from the fibroblasts of pretibial myxedema and reverse transcribed using random primers as the primers for cDNA synthesis. The resulting cDNAs were subjected to amplification by polymerase chain reaction with the use of a set of primers spanning the 5' region (+256/+275 and +616/+635) and the 3' region (+1819/+1838 and +2405/+2424) of the TSH receptor cDNA (+1 transcription start codon). They were further identified by Southern blot hybridization, with the probe spanning the 5' region (+272/+612) and the 3' region (+1908/+2268) of the TSH receptor cDNA (+ 1 transcription start codon), and sequencing. The results showed that patients with pretibial myxedema had higher titers of TSH receptor antibodies in the serum. TSH and TSH receptor antibody-binding sites were present on plasma membranes of fibroblasts derived from the skin of pretibial myxedema patients and related to the extracellular domain of the TSH receptor. These data suggest a common antigenic site in the skin and in the thyroid as a putative target for TSH receptor antibodies or lymphocytes of Graves' disease.