过表达溶质载体家族 1 成员5 和敲低慢病毒载体构建及稳定转染 RAW264.7 细胞株.

BACKGROUND: Solute carrier family 1 member 5 (SLC1A5) plays a potential role in a variety of diseases, but the exact mechanism of action is unclear. The construction of stable SLC1A5 overexpression and knockdown cell models can provide a powerful experimental tool for in-depth study of the exact role and mechanism of SLC1A5 in diseases and the discovery of potential therapeutic targets. OBJECTIVE: To construct lentiviral vectors for overexpression and knockdown of mouse SLC1A5 and establish stable transfected RAW264.7 cell lines, so as to provide an experimental foundation for further investigation of the role of SLC1A5 in inflammation. METHODS: Primers were designed and synthesized based on the SLC1A5 gene sequence, and the gene segment was amplified using polymerase chain reaction. Subsequently, the target gene segment was directionally inserted into the GV492 vector plasmid, which had been digested with AgeI/NheI enzymes, to construct recombinant lentiviral plasmids. Positive clones were further selected, and their sequences were confirmed. The pHelper1.0 plasmid vector and pHelper2.0 plasmid vector, along with the target plasmid vector, was co-cultured with 293T cells for transfection, resulting in the production and titration of lentiviral stocks. Furthermore, RAW264.7 cells were cultured in vitro, and the working concentration of puromycin was determined. Lentiviruses were separately co-cultured with RAW264.7 cells, and transfection efficiency was determined by measuring fluorescence intensity. Stable transfected cells were selected using puromycin, and real-time fluorescence quantitative PCR and western blot assay were employed to assess the gene and protein expression levels of SLC1A5 in stably transfected cell lines. RESULTS AND CONCLUSION: Sequencing results indicated a perfect match between the sequencing and target sequences, confirming the successful construction of recombinant lentiviral vectors. The titer for the overexpression SLC1A5 lentivirus was 1×109 TU/mL, while the titer for the knockdown SLC1A5 lentivirus was 3×109 TU/mL. The working concentration of puromycin for RAW264.7 cells was determined to be 3 μg/mL. The optimal conditions for transfecting RAW264.7 cells with overexpression/knockdown expression of SLC1A5 lentivirus involved the use of HiTransG P transfection enhancer with a multiplicity of infection value of 50. A significant upregulation of the gene and protein expression levels of SLC1A5 was detected in cell lines stably overexpressing SLC1A5, while gene and protein expression levels of SLC1A5 were significantly decreased in the knockdown stable cell lines. These findings indicate that lentiviral vectors for mouse SLC1A5 overexpression and knockdown have been successfully constructed and a stably transfected RAW264.7 cell line has been obtained. [ABSTRACT FROM AUTHOR]