Stable Expression of Hantavirus H8205 Strain G1/IL-2 Gene and Immune Protection of the Fusion Gene

To explore the feasibility of stable expression of Hantavirus H8205 strain G1 segment and human IL-2 fusion gene in Vero cells, and to examine the immune protection effects on mice vaccinated with this recombinant eukaryotic expression vector containing Hantavirus G1 gene and IL-2 gene. With the help of lipofectamine, the Vero cells were transfected with pcDNA3.1/HisB-IL-2-G1 and the positive cells were selected by G418. IFAT and SDS-PAGE elec- trophoresis were used to determine the stable transfection and expression of recombinant protein. Each mouse was inoculated with plasmids intramuscularly （i.m.） three times, 2 boosts were given at 2-week intervals, serum anti-hantavirus antibodies were detected by ELISA and neutralizing antibodies （NAb） were detected by Plaque Reduction Neutralization Test. The fusion protein expressed in Vero cells was 78 kD, corresponding to the estimated molecular size. The neutralizing antibody titers of mice with pcDNA3.1/HisB-IL-2-G1 were 1：20-1：80. IL-2/G1 fusion gene could be transferred in Vero cells and stably express the fusion protein. Specific humeral immune responses in mice can be induced with the recombinant eukaryotic expression vector containing the fusion gene, which lays the foundation for further development of therapeutic HTNV vaccine.

Quantitative Expression of Heat Flow versus Tectonic Deformation in the China Continent: The Effects of Plastic-Flow Network and Stable Block

Based on the heat flow data published in 1990 and 2001, a study of the factors influencing the terrestrial heat flow distribution in the China continent and its quantitative expression is carried out using the ＂Netlike Plastic-Flow＂ continental dynamics model and the methods of statistic analysis and optimum fitting. The result indicates that the factors influencing the heat flow distribution is classified into two groups, i.e. background and tectonic ones, in which the former mainly involves the non- uniform distribution of mantle heat flow, heat production of radioactive dements in the crust, heattransfer media and hydrothermal circulation, while the latter mainly involves plastic-flow networks and relatively-stable blocks. The plastic-flow network is a manifestation of shear localization in the netlike plastic-flow process in the lower lithosphere, which is composed of two sets of plastic-flow belts （PFBs） intersecting each other and, as one of the basic action regimes, controls the intraplate tectonic deformation. Relatively stable blocks （RSBs）, which are the tectonic units with relatively-high viscosities existing in the netlike plastic-flow field, as one of the principal origins, result in the development of large-seale compressional basins. PFB and RSB, as the active and quiet states of tectonic deformation, give rise to the higher and lower heat flow values, respectivdy. The provincial average heat flow in continent can be estimated using the expression qav = q0 ＋ a Pbt-c Pbk, where the three terms of the right side are background heat flow, PFB-positive contribution and RSB-negative contribution, Pbt and Pbk are the PFB- and RSB-coverage ratios, respectively, a is the coefficient of PFB- positive contribution depending mainly on the strain in the lower lithosphere, and c is the coefficient of RSB-negative contribution related mainly to the thickness of the lithosphere, the aseismic-area ratio and the tectonic age. For the major portion of the China continent excluding some of the southeastern region of China, the confidence interval of the provincial average background heat flow is qo=57.25±24.8 mW/m^2 and the PFB-positive- and RSB-negative-contribution coefficients are a=14.8-71.9 mW/m^2 and c=0-25.6 mW/m^2, respectively. The concepts of PFB and RSB effects and the heat flow expression suggested provide a new choice of the approach to the quantitative description of the characteristics of heat flow distribution in continent and their physical mechanisms.

Delivering DNA into Plant Cell by Gene Carriers of ZnS Nanoparticles

The development of nanotechnology provides a new method for genetic engineering.However,the nanoparticles as gene carriers have been mainly used in the mammalian cells so far.We observed that ZnS nanoparticles modified with positively charged poly-L-lysine（PLL） successfully delivered GUS-encoding plasmid DNA into tobacco cells by means of ultrasound-assisted method.Polymerase chain reaction（PCR） detection,Southern blot analysis and GUS histochemical staining were carried out for the regenerated plants.The stable genetic modified plants mediated by ZnS nanoparticles can be obtained.This article demonstrates the great potential of nanoparticles as gene carrier in plant transformation and proves a novel approach for plant genetic decoration.

Sustained high level transgene expression in mammalian cells mediated by the optimized piggyBac transposon system

Sustained,high level transgene expression in mammalian cells is desired in many cases for studying gene functions.Traditionally,stable transgene expression has been accomplished by using retroviral or lentiviral vectors.However,such viral vector-mediated transgene expression is often at low levels and can be reduced over time due to low copy numbers and/or chromatin remodeling repression.The piggyBac transposon has emerged as a promising nonviral vector system for efficient gene transfer into mammalian cells.Despite its inherent advantages over lentiviral and retroviral systems,piggyBac system has not been widely used,at least in part due to their limited manipulation flexibilities.Here,we seek to optimize piggyBac-mediated transgene expression and generate a more efficient,user-friendly piggyBac system.By engineering a panel of versatile piggyBac vectors and constructing recombinant adenoviruses expressing piggyBac transposase(PBase),we demonstrate that adenovirusmediated PBase expression significantly enhances the integration efficiency and expression level of transgenes in mesenchymal stem cells and osteosarcoma cells,compared to that obtained from co-transfection of the CMV-PBase plasmid.We further determine the drug selection timeline to achieve optimal stable transgene expression.Moreover,we demonstrate that the transgene copy number of piggyBac-mediated integration is approximately 10 times higher than that mediated by retroviral vectors.Using the engineered tandem expression vector,we show that three transgenes can be simultaneously expressed in a single vector with high efficiency.Thus,these results strongly suggest that the optimized piggyBac system is a valuable tool for making stable cell lines with sustained,high transgene expression.

Silica Nanoparticles-mediated Stable Genetic Transformation in Nicotiana tabacum

Nanoparticles as gene carriers become popular in the mammalian cells, whereas the application of them in plant cells is still very limited. Herein lies a report on silica nanoparticles（SiNPs） modified with positively charged poly-L-lysine（PLL） successfully delivering plasmid-encoded β-glucuronidase（GUS） gene into tobacco with the help of gene gun. The stable transgenic tobacco plants mediated by SiNPs can be obtained. Furthermore, we revealed the quantity of gene and types of receptor materials could affect the expression efficiency. In comparison to conven- tional gold particles-mediated transformation, the silica nanoparticles-mediated stable genetic transformation enhances transformation efficiency, potentially overcoming transgenic silencing. Our results demonstrate the great potential of SiNPs as gene carrier in plant genetic transformation and prove a novel approach for plant genetic decoration.

Construction of a CHO cell line with site‑specific integration to stably express exogenous proteins using the CRISPR–Cas9 technique

Chinese hamster ovary(CHO)cells are widely used in biopharmaceuticals because of their high-density suspension culture,high safety,and high similarity between expressed exogenous proteins and natural proteins.However,the level of exogenous protein expression decreases with increasing culture time;this phenomenon occurs due to the recombination of foreign genes into chromosomes through random integration.The present study integrated the foreign genes into a specific chromosomal site for stable expression based on CRISPR–Cas9 technology.The results showed that the exogenous proteins enhanced green fluorescent protein(EGFP)and human serum albumin(HSA)were successfully integrated into the vicinity of base 1969647 on chromosome NW_003613638.1 of CHO-K1 cells.The obtained positive monoclonal cell lines expressed all the corresponding exogenous proteins after 60 consecutive passages,and no significant differences in expression levels were observed.This study might provide a feasible method to construct a CHO cell line with long-term stable expression of exogenous proteins.