ABSTRACT
<p><b>OBJECTIVE</b>To labele MESPU35, a embryonic stem (ES) cell line derived from C57BL/6j mouse, with enhanced green fluorescent protein (EGFP) for further application.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>We 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.</p><p><b>CONCLUSIONS</b>The 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.</p>
Subject(s)
Animals , Mice , Embryo, Mammalian , Cell Biology , Metabolism , Green Fluorescent Proteins , Luminescent Proteins , Genetics , Mice, Inbred C57BL , Stem Cells , Metabolism , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Homologous 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.</p><p><b>RESULTS</b>EGFP 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.</p><p><b>CONCLUSIONS</b>This 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.</p>
Subject(s)
Animals , Mice , Cells, Cultured , Chromosome Mapping , Embryo, Mammalian , Cell Biology , Green Fluorescent Proteins , Hypoxanthine Phosphoribosyltransferase , Genetics , Luminescent Proteins , Metabolism , Recombination, Genetic , Stem Cells , Metabolism , TransgenesABSTRACT
<p><b>OBJECTIVE</b>To study the effects of emodin on proliferation and differentiation of 3T3-L1 preadipocyte and the possible mechanism.</p><p><b>METHODS</b>Cell 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.</p><p><b>RESULTS</b>Emodin 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.</p><p><b>CONCLUSIONS</b>The 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.</p>