Retention of the developmental pluripotency in medaka embryonic stem cells after gene transfer and long-term drug selection for gene targeting in fish

Embryonic stem (ES) cells provide a unique tool for introducing random or targeted genetic alterations, because it is possible that the desired, but extremely rare recombinant genotypes can be screened by drug selection. ES cell-mediated transgenesis has so far been limited to the mouse. In the fish medaka (Oryzias latipes) several ES cell lines have been made available. Here we report the optimized conditions for gene transfer and drug selection in the medaka ES cell line MES1 as a prelude for gene targeting in fish. MES1 cells gave rise to a moderate to high transfection efficiency by the calcium phosphate co-precipitation (5%), commercial reagents Fugene (11%), GeneJuice (21%) and electroporation (>30%). Transient gene transfer and CAT reporter assay revealed that several enhancers/promoters and their combinations including CMV, RSV and ST (the SV40 virus early gene enhancer linked to the thymidine kinase promoter) were suitable regulatory sequences to drive transgene expression in the MES1 cells. We show that neo, hyg or pac conferred resistance to G418, hygromycin or puromycin for positive selection, while the HSV-tk generated sensitivity to ganciclovir for negative selection. The positive-negative selection procedure that is widely used for gene targeting in mouse ES cells was found to be effective also in MES1 cells. Importantly, we demonstrate that MES1 cells after gene transfer and long-term drug selection retained the developmental pluripotency, as they were able to undergo induced differentiation in vitro and to contribute to various tissues and organs during chimeric embryogenesis.

Investigation of Interactions between Two Monoclonal Antibodies and SARS Virus with a Label-Free Protein Array

The investigation of interactions between two kinds of monoclonal antibodies and SARS virus with a label-free protein array technique were presented in this paper. The performance consists of three parts: a surface modification for ligand immobilization/surface, a protein array fabrication with an integrated microfluidic system for patterning, packaging and liquid handling, and a protein array reader of imaging ellipsometer. This revealed the technique could be used as an immunoassay for qualitative and quantitative detection as wen as kinetic analysis of biomolecule interaction.

Detection of hepatitis B virus markers using a biosensor based on imaging ellipsometry

A biosensor based on imaging ellipsometry (BIE) has been developed and validated in 169 patients for detecting five markers of hepatitis B virus (HBV) infection. The methodology has been established to pave the way for clinical diagnosis, including ligand screening, determination of the sensitivity, set-up of cut-off values (CoVs) and comparison with other clinical methods. A matrix assay method was established for ligand screening. The CoVs of HBV markers were derived with the help of receiver operating characteristic curves. Enzyme-linked immunosorbent assay (ELISA) was the reference method. Ligands with high bioactivity were selected and sensitivities of 1 ng/mL and 1 IU/mL for hepatitis B surface antigen (HBsAg) and surface antibody (anti-HBs) were obtained respectively. The CoVs of HBsAg, anti-HBs, hepatitis B e antigen, hepatitis B e antibody and core antibody were as follows: 15%, 18%, 15%, 20% and 15%, respectively, which were the percentages over the values of corresponding ligand controls. BIE can simultaneously detect up to five markers within 1 h with results in acceptable agreement with ELISA, and thus shows a potential for diagnosing hepatitis B with high throughput.

Evolutionary implications of avian infectious Bronchitis Virus (AIBV) analysis

For developing efficient vaccines, it is essential to identify which amino acid changes are most important to the survival of the virus. We investigate the amino acid substitution features in the Avian Infectious Bronchitis Virus (AIBV) antigenic domain o

A paradigm for virus-host coevolution: Sequential counter-adaptations between endogenous and exogenous retroviruses

Endogenous retroviruses ( ERVs) are remnants of ancient retroviral infections of the host germline transmitted vertically from generation to generation. It is hypothesized that some ERVs are used by the host as restriction factors to block the infection o

Genotyping of TRIM5 locus in northern pig-tailed macaques (Macaca leonina), a primate species susceptible to Human Immunodeficiency Virus type 1 infection

Background: The pig-tailed macaques are the only Old World monkeys known to be susceptible to human immunodeficiency virus type 1 (HIV-1) infection. We have previously reported that the TRIM5-Cyclophilin A (TRIMCyp) fusion in pig-tailed macaques (Macaca n

Secondary structural analysis of the mRNA regions encoding the hemagglutinin cleavage site basic amino acids of the avian influenza virus H5N1 subtype samples

Here we report the codon bias and the mRNA secondary structural features of the hemagglutinin (HA) cleavage site basic amino acid regions of avian influenza virus H5N1 subtypes. We have developed a dynamic extended folding strategy to predict RNA secondar

Synthesis and Anti-Human Immunodeficiency Virus Type 1 Activity of (E)-N-Phenylstyryl-N-alkylacetamide Derivatives

A series of (E)-N-phenylstyryl-N-alkylacetamides, 5, were synthesized by direct reduction-acetylation of beta-arylnitroolefins, followed by N-alkylation. The title compounds were characterized by H-1-NMR, EIMS and IR analysis. All the synthesized compound

Human immunodeficiency virus-1 genotypic drug resistance among volunteer blood donors in Yunnan, China

BACKGROUND: Drug resistance profiles of human immunodeficiency virus-1 (HIV-1) in treatment-naive infections have been reported in developed countries. However, little is known in developing countries, including China, especially in treatment-naive volunt

Anti Human Immunodeficiency Virus-1 (HIV-1) Agents 3. Synthesis and in Vitro Anti-HIV-1 Activity of Some N-Arylsulfonylindoles

In order to find compounds with superior anti human immunodeficiency virus type 1 (HIV-1) activity, twelve simple N-arylsulfonylindoles (3a-1) were synthesized and preliminarily evaluated as HIV-1 inhibitors in vitro for the first time. Several compounds

Anti Human Immunodeficiency Virus-1 (HIV-1) Agents 1. Discovery of Benzyl Phenyl Ethers as New HIV-1 Inhibitors in Vitro

Ten single benzyl phenyl ethers were synthesized and evaluated as human immunodeficiency virus-1 (HIV-1) inhibitors in vitro for the first time. Among these compounds, especially 4-nitrobenzyl phenyl ether (3h) exhibited the highest anti-HIV-1 activity wi

Anti-human Immunodeficiency Virus-1 Constituents of the Bark of Poncirus trifoliata

A total of 36 compounds (1-36) were obtained from the stem bark of Poncirus trifoliata including three new prenylated flavonoids, (-)-5,4'-dihydroxy-7,8-[(3 '',4 ''-cis-dihydroxy-3 '',4 ''-dihydro)-2 '',2 ''-dimethylpyrano]-flavone (1), (-)-5,4'-dihydroxy-7,8-[(3 ''-hydroxy-4 ''-one)-2 '',2 ''-dimethylpyrano]-flavone (2), and (-)-5,4'-dihydroxy-7,8-[(cis-3 ''-hydroxy-4 ''-ethoxy-3 '',4 ''-dihydro)-2 '',2 ''-dimethylpyrano]-flavone (3). The new structures were elucidated by means of spectroscopic methods. Compounds 1-20 were evaluated for their anti-human immunodeficiency virus-1 (HIV-1) activity, in which 2 showed significant anti-HIV-1 activity with high therapeutic index (T1) of 143.65.