OBJECTIVETo labele MESPU35, a embryonic stem (ES) cell line derived from C57BL/6j mouse, with enhanced green fluorescent protein (EGFP) for further application.

METHODSThe EGFP gene was controlled by the hybrid CA promoter/enhancer (CMV enhancer/chicken beta-actin promoter/beta-actin intron) to construct the vector of the transgene, pCA-EGFP. The vector was transfected into MESPU35 by electroporation.

RESULTSWe generated EGFP expressing ES cells demonstrating normal properties. The green fluorescence of EGFP expressing cells was maintained in propagation of the ES cells for more than 30 passages as well as in differentiated cells. Cultured in suspension, the "green" ES cells aggregated, and formed embryoid bodies maintaining the green fluorescence at varying developmental stages. The "green" embryoid bodies could expand and differentiate into various types of cells, exhibiting ubiquitous green fluorescence.

CONCLUSIONSThe hybrid CA promoter/enhancer used to control the EGFP expressing ES cells, resulted in more intense and ubiquitous activity. The EGFP transfected cells yield bright green fluorescence, which can be visualized in real time and in situ. In addition, the ES cells, MESPU35, are derived from C57BL/6j mice, which are the most widely used in oncology, physiology and genetics. Compared to 129 substrains, C57BL/6j mice avoid a number of potential problems apparent in the other strains.

Animals ; Embryo, Mammalian ; cytology ; metabolism ; Green Fluorescent Proteins ; Luminescent Proteins ; genetics ; Mice ; Mice, Inbred C57BL ; Stem Cells ; metabolism ; Transfection

OBJECTIVETo study the effects of emodin on proliferation and differentiation of 3T3-L1 preadipocyte and the possible mechanism.

METHODSCell proliferation was determined by MTT spectrophotometry, cell differentiation was determined by Oil Red O staining,and fatty acid synthase (FAS) activity was determined by spectrophotometry.

RESULTSEmodin promoted proliferation of 3T3-L1 preadipocyte at low concentration and inhibited the proliferation at high concentration in a dose-related manner. In contrast, it inhibited cell differentiation into adipocyte at low concentration in a dose-related manner. In vitro emodin inhibited the activity of FAS in a dose-related manner.

CONCLUSIONSThe effects of emodin on 3T3-L1 cell's proliferation and differentiation are dose dependent. Emodin inhibits the activity of FAS. Our results suggest that emodin should have a potential to serve as a fat-reducing drug.

3T3 Cells ; Adipocytes ; drug effects ; physiology ; Animals ; Cell Differentiation ; drug effects ; Cell Division ; drug effects ; Emodin ; pharmacology ; Fatty Acid Synthases ; antagonists & inhibitors ; Lipid Metabolism ; Mice ; Stem Cells ; drug effects ; physiology

OBJECTIVETo label embryonic stem (ES) cells with enhanced green fluorescent protein (EGFP) on the hypoxanthineguanine phosphoribosyl transferase (HPRT) gene locus for the first time to provide a convenient and efficient way for cell tracking and manipulation in the studies of transplantation and stem cell therapy.

METHODSHomologous fragments were obtained by polymerase chain reaction (PCR), from which the gene targeting vector pHPRT-EGFP was constructed. The linearized vector was introduced into ES cells by electroporation. The G418(r)6TG(r) cell clones were obtained after selection with G418 and 6TG media. The integration patterns of these resistant cell clones were identified with Southern blotting.

RESULTSEGFP expressing ES cells on the locus of HPRT were successfully generated. They have normal properties, such as karyotype, viability and differentiation ability. The green fluorescence of EGFP expressing cells was maintained in propagation of the ES cells for more than 30 passages and in differentiated cells. Cultured in suspension, the "green" ES cells aggregated and formed embryoid bodies, retaining the green fluorescence at varying developmental stages. The "green" embryoid bodies could expand and differentiate into various types of cells, exhibiting ubiquitous green fluorescence.

CONCLUSIONSThis generation of "green" targeted ES cells is described in an efficient protocol for obtaining the homologous fragments by PCR. Introducing the marker gene in the genome of ES cells, we should be able to manipulate them in vitro and use them as vehicles in cell-replacement therapy as well as for other biomedical and research purposes.

Animals ; Cells, Cultured ; Chromosome Mapping ; Embryo, Mammalian ; cytology ; Green Fluorescent Proteins ; Hypoxanthine Phosphoribosyltransferase ; genetics ; Luminescent Proteins ; metabolism ; Mice ; Recombination, Genetic ; Stem Cells ; metabolism ; Transgenes

Objective: To monitor and analyze the epidemiological characteristics of influenza in schools and understand the disease burden of students, and to provide a scientific reference for instructing the prevention of influenza in schools. Methods: A school influenza surveillance sentinel to conduct influenza like case (ILI) surveillance and outbreak surveillance. Through network, we understood the burden of flu disease among students. Descriptive epidemiology was used to analyze influenza like case surveillance and questionnaire survey data. Results: Surveillance confirmed that from the 42th week of 2019 to the 1st week of 2020, the cumulative reported ILI of 3 school influenza surveillance sites in Jinan accounted for 7.91% (ILI%) of the total number of surveillance personnel during the same period, with the highest ILI% (24.19%) of kindergarten children, ILI% gradually decreased with the increase of grade, and teachers were the lowest. The reporting of ILI was concentrated in the 49th to 52nd week of 2019, during which the reported influenza like cases accounted for 84.81% of the total number of ILI reported during the surveillance period. Two influenza outbreaks were monitored. The pathogens were H3N2 and B (Victoria). The epidemics mainly occurred in the lower grades of elementary school. A survey of 2 297 students found that 577 people had fever and respiratory symptoms since October 2019. Among them, 85.26% of them went to the hospital, 32.75% of those who used anti influenza drugs such as oseltamivir, and 64.81% of those who used antibiotics. 42.63% received infusion therapy, 3.99% were hospitalized, and the average cost of inpatients was 6 686 yuan. The sick students were absent from school for an average of 3.77 days, and the parents of the sick children missed work for an average of 4.26 days. Conclusion Surveillance of influenza like cases in schools is an important way to proactively discover influenza epidemic trends and outbreaks, and to accurately grasp the characteristics of influenza epidemics in schools. The key populations affected by influenza are kindergarten children and lower grades of primary school students. Suffering from influenza has caused a heavy disease burden on students and children in kindergartens, and is also an important factor that causes student absenteeism and parents to miss work.