Construction of lentiviral vectors for solute carrier family 1 member 5  overexpression and knockdown and stably transfected RAW264.7 cell line

doi:10.12307/2025.025

Abstract

Abstract: 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: (1) Sequencing results indicated a perfect match between the sequencing and target sequences, confirming the successful construction of recombinant lentiviral vectors. (2) 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. (3) The working concentration of puromycin for RAW264.7 cells was determined to be 3 μg/mL. (4) 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. (5) 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.

Key words: lentiviral vector, solute carrier family 1 member 5, SLC1A5, overexpression, knockdown, RAW264.7 cell, stably transfected cell line

CLC Number:

Guo Daxin, Fan Susu, Zhu Zhendong, Hou Jianhong, Zhang Xuan. Construction of lentiviral vectors for solute carrier family 1 member 5 overexpression and knockdown and stably transfected RAW264.7 cell line[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(7): 1414-1421.

Figures/Tables 3

2.1 慢病毒载体构建及质粒鉴定 SLC1A5过表达/敲低慢病毒载体GV492 和GV493见图1A，B。将鉴定出的阳性单克隆菌落37 ℃培养18 h，取适量菌液测序，并与目的基因的反向互补序列进行比对。化学合成目的基因序列见图2A，阳性重组克隆测序比对结果见图2B。比对结果显示与设计目的序列保持一致，说明目的基因正确插入GV492载体。 2.2 慢病毒的包装及滴度测定 SLC1A5过表达慢病毒滴度为1×109 TU/mL，见图3A，SLC1A5敲低慢病毒滴度为3×109 TU/mL，见图3B。 2.3 嘌呤霉素工作浓度的确定嘌呤霉素质量浓度的增加与RAW264.7细胞死亡呈正相关，嘌呤霉素质量浓度为3.5 μg/mL及以上时基本无细胞存活，见图4A。随着培养时间的延长，嘌呤霉素质量浓度为3.0 μg/mL时即无细胞存活，见图4B。 2.4 过表达/敲低SLC1A5慢病毒最佳转染条件在过表达SLC1A5慢病毒转染RAW264.7细胞中，当各组的感染复数为10时，只有少数细胞带有荧光，表示较少数的细胞成功转染，且荧光强度较弱；与完全培养基组和转染增强液A组相比，转染增强液P组细胞状态良好，基本无细胞死亡，荧光强度较强；当感染复数为50时，各组被转染的RAW264.7细胞数增多，荧光程度较强，转染效率达到80%左右，其中转染增强液P组转染效率较好；当感染复数为100时，各组被转染RAW264.7细胞未见明显增多，细胞生长情况一般，转染效率达到90%左右，见图5。在敲低SLC1A5慢病毒转染RAW264.7细胞中，当感染复数为10时，仅有极少数RAW264.7细胞成功转染，荧光强度较弱；当感染复数为50时，被转染RAW264.7细胞增多，荧光强度较强，细胞状态良好，无明显细胞死亡，转染效率达到80%左右；当感染复数为100时，各组被转染RAW264.7细胞未见明显增多，细胞生长情况一般，转染效率达到90%左右，见图6。该实验中最佳转染条件的选择原则：在保证细胞生长状态良好的前提下使用最少量的慢病毒以及荧光强度较强的转染增强液，基于上述原则，过表达/敲低SLC1A5慢病毒转染RAW264.7细胞的最佳转染条件皆为HiTransG P转染增强液且感染复数值等于50。 2.5 过表达SLC1A5慢病毒稳转RAW264.7细胞中SLC1A5的表达量完全培养基中加入HiTransG P及感染复数为50的过表达目的基因慢病毒与RAW264.7细胞培养72 h，含3 μg/mL嘌呤霉素的完全培养基筛选第4，5代获得SLC1A5稳定高表达的RAW264.7细胞株。荧光显微镜观察结果显示目的基因过表达RAW264.7细胞株生长状态良好，达到100%的转染效率，见图7。提取总RNA进行qPCR定量检测，与空白对照组相比，SLC1A5-H组的SLC1A5基因表达量有显著升高，差异有显著性意义(P < 0.001)，而阴性对照组与空白对照组的SLC1A5基因表达量未发生明显变化，见图8。 2.6 敲低SLC1A5慢病毒转染RAW264.7细胞中SLC1A5的表达量在完全培养基中加入含HiTransG P转染液以及感染复数为50的敲低慢病毒与RAW264.7细胞培养72 h后，使用含3 μg/mL嘌呤霉素的完全培养基构建稳定敲低RAW264.7细胞株。荧光显微镜观察结果显示稳转RAW264.7细胞株生长状态良好，达到100%的转染效率，见图9。提取总RNA进行qPCR检测，与空白对照组比较，SLC1A5-L组的SLC1A5表达量有显著降低，差异有显著性意义(P < 0.01)，阴性对照组与空白对照组的SLC1A5表达量未出现明显变化，见图10。 2.7 过表达/敲低SLC1A5慢病毒转染RAW264.7细胞中SLC1A5的相对蛋白表达水平经Image J灰度统计分析空白对照组、阴性对照组、SLC1A5-L组、SLC1A5-H组的SLC1A5蛋白相对表达水平，与阴性对照组相比，SLC1A5-L组细胞中SLC1A5蛋白表达量明显降低(P < 0.05)；与阴性对照组相比，SLC1A5-H组细胞中SLC1A5蛋白表达量明显增高(P < 0.001)。因此在蛋白表达水平上，经过表达/敲低SLC1A5慢病毒转染RAW264.7细胞也得到了明显增高或降低，见图11。 "

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