Objective To optimize and establish the methodology for culturing and inducing differentiation of mouse preadipocytes 3T3-L1. Methods The mouse cells 3T3-L1 were incubated in DMEM medium contained with 10%FBS, during which the incubation medium was refreshed every 2 to 3 days. Two methods were used to introduce differentiation, including three-drug combination group and four-drug combination group. The protocol of mediumⅠin three-drug combination group including insulin 10 mg/L, IBMX 0.5 mmol/L and DEX 1.0μmol/L. The protocol of mediumⅠin four-drug combination group including indometacin 0.1 mmol/L based on those of three-drug combination group. Both of them were incubated for 2 days and continuous for 2 times. And medium Ⅱ included insulin 10 mg/L for 2-day culturing and continuous for 2 times. Oil red O staining was used to observe the morphological changes of two groups of cells before and after treatment under inverted microscope. Results Mouse preadipocytes 3T3-L1 appeared in good conditions and grew in a paving stone fashion. These cells covered homogeneously the bottom of incubators, the culture medium refreshed every 2 days. The results of four-drug combination group were better than those of three-drug combination group. After three-drug combination induced differentiation, there was no significant change in cell morphology. Comparing with three-drug combination induced differentiation, four-drug combination was successfully achieved in over 90% of the cell inducing, which were round-shaped, with jacinth ester droplets by oil-red O staining. Conclusion We have optimized the method for culturing and inducing differentiation of mouse preadipocytes 3T3-L1 by adding indometacin on the basis of the three-drug combination induced differentiation.

Rabbit hemorrhagic disease (RHD) is contagious and highly lethal. Commercial vaccines against RHD are produced from the livers of experimentally infected rabbits. Although several groups have reported that recombinant subunit vaccines against rabbit hemorrhagic disease virus (RHDV) are promising, application of the vaccines has been restricted due to high production costs or low yield. In the present study, we performed codon optimization of the capsid gene to increase the number of preference codons and eliminate rare codons in Spodoptera frugiperda 9 (Sf9) cells. The capsid gene was then subcloned into the pFastBac plasmid, and the recombinant baculoviruses were identified with a plaque assay. As expected, expression of the optimized capsid protein was markedly increased in the Sf9 cells, and the recombinant capsid proteins self-assembled into virus-like particles (VLPs) that were released into the cell supernatant. Rabbits inoculated with the supernatant and the purified VLPs were protected against RHDV challenge. A rapid, specific antibody response against RHDV was detected by an ELISA in all of the experimental groups. In conclusion, this strategy of producing a recombinant subunit vaccine antigen can be used to develop a low-cost, insect cell-derived recombinant subunit vaccine against RHDV.
Animals ; Antigens, Viral/genetics/metabolism ; Caliciviridae Infections/prevention & control/*veterinary/virology ; Capsid Proteins/*genetics/metabolism ; Cell Culture Techniques/*methods ; Codon/genetics/metabolism ; Enzyme-Linked Immunosorbent Assay/veterinary ; *Gene Expression Regulation, Viral ; Hemorrhagic Disease Virus, Rabbit/*genetics/immunology ; *Rabbits ; Recombinant Proteins/genetics/metabolism ; Sf9 Cells ; Spodoptera ; Viral Structural Proteins/*genetics/metabolism ; Viral Vaccines/genetics/immunology

T-cell internal antigen-1 (TIA-1) has roles in regulating alternative pre-mRNA splicing, mRNA translation, and stress granule (SG) formation in human cells. As an evolutionarily conserved response to environmental stress, SGs have been reported in various species. However, SG formation in chicken cells and the role of chicken TIA-1 (cTIA-1) in SG assembly has not been elucidated. In the present study, we cloned cTIA-1 and showed that it facilitates the assembly of canonical SGs in both human and chicken cells. Overexpression of the chicken prion-related domain (cPRD) of cTIA-1 that bore an N-terminal green fluorescent protein (GFP) tag (pntGFP-cPRD) or Flag tag (pFlag-cPRD) induced the production of typical SGs. However, C-terminal GFP-tagged cPRD induced notably large cytoplasmic granules that were devoid of endogenous G3BP1 and remained stable when exposed to cycloheximide, indicating that these were not typical SGs, and that the pntGFP tag influences cPRD localization. Finally, endogenous cTIA-1 was recruited to SGs in chicken cells and tissues under environmental stress. Taken together, our study provide evidence that cTIA-1 has a role in canonical SG formation in chicken cells and tissues. Our results also indicate that cPRD is necessary for SG aggregation.
Chickens ; Clone Cells ; Cycloheximide ; Cytoplasmic Granules ; Humans ; Protein Biosynthesis ; RNA Precursors ; RNA-Binding Proteins ; T-Lymphocytes

To construct a eukaryotic expression plasmid containing the luciferase reporter gene (Fluc) to quickly detect apoptosis. Four amino acids, Asp-Glu-Val-Asp (DEVD), the recognize motif of Caspase-3, were introduced into the middle of the Fluc-C and N fragment. Meanwhile, four amino acids, Asp-Glu-Val-Gly (DEVG), were selected as a negative control. Subsequently, the recombinant gene was cloned into the N and C terminal end of the split intein, and named as pFluc-DEVD and pFluc-DEVG. Then the plasmids were transfected into cells and renilla luciferase was co-transfected in each sample as an internal control for transfection efficiency. Then the apoptosis level was detected by the double luciferase reporter gene and the Western blotting analysis. The results showed that when apoptosis occurred, the content of firefly luciferase expressed in the pFluc-DEVD plasmid transfected group was about 3 times higher than pFluc-DEVG plasmid transfected group. Furthermore, Western blotting detection indicated that the Fluc level was significantly increased in pFluc-DEVD transfected group when pre-treated by apoptosis stimulants. The activation degree of Caspase-3 was closely related to the expression of Fluc, and had a significant statistical difference. These results confirmed that firefly luciferase protein expressed by pFluc-DEVD plasmid can be cleaved by the intracellular Caspase-3 enzyme, and this plasmid can accurately reflect the cell apoptosis level, which provides a useful method for quantitative detection of apoptosis.
Apoptosis ; Genes, Reporter ; Luciferases, Firefly ; Transfection