Long-term replication of Epstein-Barr virus-derived episomal vectors in the rodent cells

AbstractPlasmids containing the origin of replication, oriP, of the Epstein-Barr virus (EBV) and EBV nuclear antigen-1 genes replicate extrachromosomally in primate cells. However, these plasmids have been believed not to replicate in rodent cells. We demonstrate here that these plasmids can replicate in some types of rodent cells over a long period. This result should offer not only the new insight into the mechanisms of species-specific replication of EBV, but also the possibility that an EBV-based vector can be used for gene transfer experiments in non-primate cells and an animal experiment regarding human gene therapy

A trial of somatic gene targeting in vivo with an adenovirus vector

BACKGROUND: Gene targeting in vivo provides a potentially powerful method for gene analysis and gene therapy. In order to sensitively detect and accurately measure designed sequence changes, we have used a transgenic mouse system, MutaMouse, which has been developed for detection of mutation in vivo. It carries bacteriophage lambda genome with lacZ(+ )gene, whose change to lacZ-negative allele is detected after in vitro packaging into bacteriophage particles. We have also demonstrated that gene transfer with a replication-defective adenovirus vector can achieve efficient and accurate gene targeting in vitro. METHODS: An 8 kb long DNA corresponding to the bacteriophage lambda transgene with one of two lacZ-negative single-base-pair-substitution mutant allele was inserted into a replication-defective adenovirus vector. This recombinant adenovirus was injected to the transgenic mice via tail-vein. Twenty-four hours later, genomic DNA was extracted from the liver tissue and the lambda::lacZ were recovered by in vitro packaging. The lacZ-negative phage was detected as a plaque former on agar with phenyl-beta-D-galactoside. RESULTS: The mutant frequency of the lacZ-negative recombinant adenovirus injected mice was at the same level with the control mouse (~1/10000). Our further restriction analysis did not detect any designed recombinant. CONCLUSION: The frequency of gene targeting in the mouse liver by these recombinant adenoviruses was shown to be less than 1/20000 in our assay. However, these results will aid the development of a sensitive, reliable and PCR-independent assay for gene targeting in vivo mediated by virus vectors and other means

Mossbauer studies on FeSe and FeTe

We carried out 57Fe Mossbauer measurements for FeSe and Fe1.08Te to investigate the magnetic properties. There was no sign of magnetic ordering above 4.2 K for superconducting FeSe. The magnetic sextet corresponding to antiferromagnetic ordering of Fe in low-spin state was observed for non-superconducting Fe1.08Te.Comment: 5 pages, 2 figures, proceedings of M2S conferenc

Long-Term Self-Renewal of Human ES/iPS-Derived Hepatoblast-like Cells on Human Laminin 111-Coated Dishes

SummaryThe establishment of self-renewing hepatoblast-like cells (HBCs) from human pluripotent stem cells (PSCs) would realize a stable supply of hepatocyte-like cells for medical applications. However, the functional characterization of human PSC-derived HBCs was not enough. To purify and expand human PSC-derived HBCs, human PSC-derived HBCs were cultured on dishes coated with various types of human recombinant laminins (LN). Human PSC-derived HBCs attached to human laminin-111 (LN111)-coated dish via integrin alpha 6 and beta 1 and were purified and expanded by culturing on the LN111-coated dish, but not by culturing on dishes coated with other laminin isoforms. By culturing on the LN111-coated dish, human PSC-derived HBCs were maintained for more than 3 months and had the ability to differentiate into both hepatocyte-like cells and cholangiocyte-like cells. These expandable human PSC-derived HBCs would be manageable tools for drug screening, experimental platforms to elucidate mechanisms of hepatoblasts, and cell sources for hepatic regenerative therapy

Molecular dynamics study on DNA damage by tritium disintegration

Using molecular dynamics (MD) simulation, we simulate the structural change of a telomeric DNA by β-decay of substituted tritium to helium-3. The configuration of the telomeric DNA is obtained by removing TRF2 protein from the TRF2-Dbd-DNA complex (Protein Data Bank ID is 3SJM). We assume that hydrogens (H) of guanines in the telomeric DNA are replaced to helium-3. Since this replacement of the H atoms to the 3He atoms changes the charge distribution significantly, the charge distribution used in the MD simulation for the modified guanine is obtained by the density functional theory calculations. We adopt, as the MD simulation, nanoscale molecular dynamics code with CHARMM36 force field using Langevin thermostat and Nosé–Hoover Langevin piston to control the temperature and pressure of the system, respectively. Moreover, changing both the number of replaced guanine N and the temperature of the system T, we calculate the root mean square deviation RMSD to quantify the dependence of the durability of the telomeric DNA on the β-decays. From the MD simulation, it is found that as N or T becomes larger, the RMSD of the DNA becomes also larger. Namely, it denotes that as the intensity of the β-decays becomes larger or as the temperature is increased, the DNA structure becomes more fragile

Efficient and Directive Generation of Two Distinct Endoderm Lineages from Human ESCs and iPSCs by Differentiation Stage-Specific SOX17 Transduction

The establishment of methods for directive differentiation from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is important for regenerative medicine. Although Sry-related HMG box 17 (SOX17) overexpression in ESCs leads to differentiation of either extraembryonic or definitive endoderm cells, respectively, the mechanism of these distinct results remains unknown. Therefore, we utilized a transient adenovirus vector-mediated overexpression system to mimic the SOX17 expression pattern of embryogenesis. The number of alpha-fetoprotein-positive extraembryonic endoderm (ExEn) cells was increased by transient SOX17 transduction in human ESC- and iPSC-derived primitive endoderm cells. In contrast, the number of hematopoietically expressed homeobox (HEX)-positive definitive endoderm (DE) cells, which correspond to the anterior DE in vivo, was increased by transient adenovirus vector-mediated SOX17 expression in human ESC- and iPSC-derived mesendoderm cells. Moreover, hepatocyte-like cells were efficiently generated by sequential transduction of SOX17 and HEX. Our findings show that a stage-specific transduction of SOX17 in the primitive endoderm or mesendoderm promotes directive ExEn or DE differentiation by SOX17 transduction, respectively