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Updated: Aug 8, 2026
A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells
Published on: June 16, 2022
[Construction of the p-mfgl2shRNA and its effect on mfgl2 expression in vitro]
Zhi-mo Wang1, Wei-ming Yan, Dong Xi
1Laboratory of Infectious Immunology and Department of Infectious Diseases, Tongji Hospital of Tongji Medical College, Hua Zhong University of Science and Technology, Wuhan 430030, China.
Objective:
To construct the siRNA plasmid for mfgl2 gene, which has been reported to be involved in a variety of disease developments including fulminant viral hepatitis, acute rejection of allo/zero transplantation and fetal loss syndrome, and to investigate its inhibitory effects on mfgl2 expression in vitro.
Methods:
A plasmid p-mfgl2shRNA complimentary to the sequence responsible for the functional domain of mouse fgl2 (mfgl2) was constructed. The pcDNA3.1 mfgl2 expression construct was able to show a satisfactory fgl2 protein expression. The plasmid expression pEGFP and a construct expressing irrelevant shRNA with a random combination of the p-mfgl2shRNA sequence were used as controls. A pEGFP-mfgl2 expressing mfgl2-EGFP fusion protein was also constructed for screening of the effect of p-mfgl2shRNA on the mfgl2 expression.
Results:
Cotransfection of p-mfgl2shRNA with pEGFP-mfgl2 decreased green fluorescent cells and the lightness of fluorescence within the cells at the 24 h, 48 h and 72 h post-transfection when compared with that in the control groups which were solely transfected with pEGFP-mfgl2. Furthermore the mfgl2 expression was significantly reduced when the pcDNA3.1 mfgl2 expression construct was cotransfected with p-mfgl2shRNA both at mRNA level by RT-PCR and protein level by RT-PCR, immunohistochemistry staining and FACS in both CHO cell and Hela cell lines.
Conclusions:
The study demonstrated that the construct of p-mfgl2shRNA successfully interfered in the mfgl2 expression in vitro. It provides a basis for a further investigation of effect in vivo.
