Production of Transgenic Pigs Mediated by Pseudotyped Lentivirus and Sperm

Sperm-mediated gene transfer can be a very efficient method to produce transgenic pigs, however, the results from different laboratories had not been widely repeated. Genomic integration of transgene by injection of pseudotyped lentivirus to the perivitelline space has been proved to be a reliable route to generate transgenic animals. To test whether transgene in the lentivirus can be delivered by sperm, we studied incubation of pseudotyped lentiviruses and sperm before insemination. After incubation with pig spermatozoa, 62±3 lentiviral particles were detected per 100 sperm cells using quantitative real-time RT-PCR. The association of lentivirus with sperm was further confirmed by electron microscopy. The sperm incubated with lentiviral particles were artificially inseminated into pigs. Of the 59 piglets born from inseminated 5 sows, 6 piglets (10.17%) carried the transgene based on the PCR identification. Foreign gene and EGFP was successfully detected in ear tissue biopsies from two PCR-positive pigs, revealed via in situ hybridization and immunohistochemistry. Offspring of one PCR-positive boar with normal sows showed PCR-positive. Two PCR-positive founders and offsprings of PCR-positive boar were further identified by Southern-blot analysis, out of which the two founders and two offsprings were positive in Southern blotting, strongly indicating integration of foreign gene into genome. The results indicate that incubation of sperm with pseudotyped lentiviruses can incorporated with sperm-mediated gene transfer to produce transgenic pigs with improved efficiency

Analysis on vibration of propeller with unilateral edge trimming based on parametric modeling

Objective In engineering, the edge trimming method is often used to solve the problem of ship-machine-propeller mismatch caused by excessive load. In order to explore the effects of edge trimming on the vibration characteristics of the propeller, this paper studies the vibration of a propeller with edge trimming based on parametric modeling. MethodsDTMB4381, DTMB4382 and DTMB4383 propellers are selected as research examples, then Matlab programming technology and the ANSYS secondary development program are combined to obtain the influence laws of different unilateral edge trimming on the vibration characteristics of the propeller, and the different reasons behind the corresponding influence laws are analyzed. ResultsFor the DTMB4381 propeller without skew, the natural frequency increases slightly with the increase of the edge trimming amount, but the mode shape does not change much. For the DTMB4382 and DTMB4383 propellers with skew, with the increase of the edge trimming amount, the change law of the natural frequency remains basically the same as that of the DTMB4381 propeller without skew. ConclusionsSkew does not change the vibration characteristics of a propeller under different unilateral trimming amounts; with the increase of the trimming amount, the natural frequency change rate of the two types of propeller shapes after chamfering will be significantly improved, and the increase is more pronounced at higher levels. The corresponding secondary development program can help the relevant personnel to select the optimal trimming scheme from the perspective of vibration characteristics in practical engineering, providing a theoretical basis and guidance for the design and improvement of trimming schemes

Aberrant Regulation of mRNA m6A Modification in Cancer Development

N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic messenger RNAs (mRNAs). The m6A modification in RNA can be catalyzed by methyltransferases, or removed by demethylases, which are termed m6A writers and erasers, respectively. Selective recognition and binding by distinct m6A reader proteins lead mRNA to divergent destinies. m6A has been reported to influence almost every stage of mRNA metabolism and to regulate multiple biological processes. Accumulating evidence strongly supports the correlation between aberrant cellular m6A level and cancer. We summarize here that deregulation of m6A modification, resulting from aberrant expression or function of m6A writers, erasers, readers or some other protein factors, is associated with carcinogenesis and cancer progression. Understanding the regulation and functional mechanism of mRNA m6A modification in cancer development may help in developing novel and efficient strategies for the diagnosis, prognosis and treatment of human cancers

A Plant-Produced Antigen Elicits Potent Immune Responses against West Nile Virus in Mice

We described the rapid production of the domain III (DIII) of the envelope (E) protein in plants as a vaccine candidate for West Nile Virus (WNV). Using various combinations of vector modules of a deconstructed viral vector expression system, DIII was produced in three subcellular compartments in leaves of Nicotiana benthamiana by transient expression. DIII expressed at much higher levels when targeted to the endoplasmic reticulum (ER) than that targeted to the chloroplast or the cytosol, with accumulation level up to 73 μg DIII per gram of leaf fresh weight within 4 days after infiltration. Plant ER-derived DIII was soluble and readily purified to > 95% homogeneity without the time-consuming process of denaturing and refolding. Further analysis revealed that plant-produced DIII was processed properly and demonstrated specific binding to an anti-DIII monoclonal antibody that recognizes a conformational epitope. Furthermore, subcutaneous immunization of mice with 5 and 25 μg of purified DIII elicited a potent systemic response. This study provided the proof of principle for rapidly producing immunogenic vaccine candidates against WNV in plants with low cost and scalability

A plant-produced antigen elicits potent immune responses against west nile virus in mice,”

We described the rapid production of the domain III (DIII) of the envelope (E) protein in plants as a vaccine candidate for West Nile Virus (WNV). Using various combinations of vector modules of a deconstructed viral vector expression system, DIII was produced in three subcellular compartments in leaves of Nicotiana benthamiana by transient expression. DIII expressed at much higher levels when targeted to the endoplasmic reticulum (ER) than that targeted to the chloroplast or the cytosol, with accumulation level up to 73 g DIII per gram of leaf fresh weight within 4 days after infiltration. Plant ER-derived DIII was soluble and readily purified to > 95% homogeneity without the time-consuming process of denaturing and refolding. Further analysis revealed that plantproduced DIII was processed properly and demonstrated specific binding to an anti-DIII monoclonal antibody that recognizes a conformational epitope. Furthermore, subcutaneous immunization of mice with 5 and 25 g of purified DIII elicited a potent systemic response. This study provided the proof of principle for rapidly producing immunogenic vaccine candidates against WNV in plants with low cost and scalability

Transportation characteristics of δ 13C in the plants-soil-bedrock-cave system in Chongqing karst area

Here we use an analytical method to determine δ 13C in local plants and organic matter in the soils above Furong cave, Chongqing, China. We also monitored δ 13C in dissolved inorganic carbon (DIC) of drip water, δ 13C of active deposits under the drip waters, and the concentration of air CO2 (PCO2). Based on these, we preliminarily studied the transportation characteristics of stable carbon isotope (13C) in cave system of the subtropical karst area. The average δ 13C value of 27 local plant samples, which belong to 16 families, was −32‰ and the weighted δ 13C for surface dry biomass was −33‰. We found that for 54 soil samples collected from 5 soil profiles, δ 13C of soil organic matters was −22‰, which could be attributed to the different transportation rates of stable carbon isotopes during the decomposition of plants and organic matters in soils. The relatively lighter 12C tended to transfer into gaseous CO2, which made the relatively heavier 13C concentrated in the soils. On the basis of monitoring of DIC-δ 13C in drip waters from July 2009 to June 2010, we found that values in winter months were heavier and values in summer months were lighter in general, the reason of which was that in summer months, both the temperature and the humidity were comparatively higher, resulted in more CO2 with lighter δ 13C generated from organic matters decomposition and plants respiration. The average DIC-δ 13C value was −11‰, about 11‰ heavier than the δ 13C of organic matters in soils, which proved that part of DIC in cave drip water was sourced from dissolution of inorganic carbonate (host rock, with heavier δ 13C). As for the δ 13C of active deposits at five drip water sites in Furong cave, they had almost the same variation with relatively light values. In other words, these active speleothems were deposited at equilibrium conditions for isotopic fractionation. These results suggest that the carbon isotopic information of speleothems could be used to track the evolution of local vegetation in certain situations