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Objective: The purpose of the article is to evaluate the changes in lipid metabolism in bovine mammary-gland epithelial MAC-T cells after PKM2 knockdown.Results: MAC-T cells stably expressing low levels of PKM2 were established with lentivirus-mediated small hairpin RNA. Although the knockdown of PKM2 had no effect on MAC-T cell growth, the reduced expression of PKM2 attenuated the mRNA and protein expression of key enzymes involved in sterol synthesis through the SREBP pathway.Conclusions: The downregulation of PKM2 significantly influenced lipid synthesis in bovine mammary-gland epithelial MAC-T cells. These findings extend our understanding of the crosstalk between glycolysis and lipid metabolism in bovine mammary-gland epithelial cells. [ABSTRACT FROM AUTHOR]

Objectives: To evaluate four 5′-UTRs on GFP expression in HEK293T cells. Results: The recombinant plasmids were constructed by restriction enzyme digestion, digestion and DNA sequencing. Quantitative real-time PCR and western blotting results showed that the transcription and translation level of PPRV-GFP mRNA was significantly lower than that of the other reporters. The transcription and translation level of ChEF1-GFP was the highest in HEK293T cells. Conclusions: Different UTRs can significantly affect protein expression. Additionally, the findings also will be useful in biological applications that require tuning of gene expression and system optimization. [ABSTRACT FROM AUTHOR]

Objectives: To determine the effects of the Sus scrofa matrix attachment region (SusMAR) on transgene expression in HEK293T cells. Results: Three expression vectors with the MAR at different sites in the PiggyBac (PB) transposon vector backbone were compared: two MARs flanking the β-galactosidase (β-gal) expression cassette, and one at the upstream or downstream site. Bos taurus MAR (BosMAR) and a β-gal expression cassette without MARs were the positive and negative controls, respectively. Compared to the control, β-gal activity of all SusMAR and BosMAR vectors was significantly improved in the presence of PB transposase (PBase). However, only the downstream SusMAR and upstream BosMAR vectors showed increased expression in the absence of PBase. Expression was significantly increased in all vectors with the PBase group compared to those without the PBase group. Gene copy numbers were not increased compared to the negative control. Conclusions: SusMAR enhanced recombinant gene expression levels and stability in HEK293T cells, was not increase transgene copy number. These results could facilitate the development of vectors for stable production of therapeutic proteins. [ABSTRACT FROM AUTHOR]

EP0 is an important early gene that modulates the life cycle of pseudorabies virus (PRV). A guanine-rich sequence overlapping with three Sp1 binding sites is located upstream of the transcription start site (TSS) in the EP0 promoter. Using native polyacrylamide gel electrophoresis (PAGE) and circular dichroism (CD), we verified that the G-rich region in the EP0 promoter forms an intramolecular parallel G-quadruplex (G4) in the presence of K+ ions. Further dimethyl sulphate (DMS) footprinting and Taq polymerase stop assays indicates the potential polymorphic folding of G4. In addition, a small chemical ligand, pyridostatin (PDS), promotes and stabilizes the formation of G4. Interestingly, based on the results of electrophoretic mobility shift assays (EMSA), the Sp1 protein bound to G4-bearing DNA with more affinity than DNA lacking the G4 structure. According to the luciferase reporter assay, G4 negatively regulates the EP0 promoter activity. These results demonstrate that Sp1 and G4 cooperate to regulate EP0 promoter activity. [ABSTRACT FROM AUTHOR]