Related Experiment Video
Updated: May 7, 2026
Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy
Published on: April 3, 2018
Antiapoptotic gene BAG-1 vector structure of RNA interference and endogenous targeted screening in colon cancer cell
Nian-feng Sun1, Ai-ling Tian, Zhan-ao Liu
1Department of General Surgery, Qilu Hospital of Shandong University, Jinan, 250012, China.
Abstract:
The purposes of the present work were to construct the shRNA plasmids for BAG-1 gene of human and test the expression of mRNA and protein of BAG-1. Recombinant plasmids containing green fluorescent protein reporter genes are constructed using gene cloning methods. The shRNA plasmids for the BAG-1 gene are constructed by RNA interference technology. We applied fluorescent plasmid-transfected target cells in the cell transfection experiments and monitored the transfection rate of plasmids by observing the fluorescence amount. We transfected three synthesized shRNA in target screening cell and adopted RT-PCR and Western test to identify the difference of target gene transfection and translation level in cells. The specific shRNA plasmid for the BAG-1 gene was successfully recombined, and stably transfected colon cancer Lo Vo cell lines were obtained. The results present that the constructed shRNA plasmids significantly inhibited the expression of mRNA and protein of Lo Vo cell BAG-1, and can maintain the effect for a long term. pGPH1/GFP/Neo-BAG-1-homo-825 was screened as the optimum sequence of interference so as to lay a solid foundation to explore into the research on the BAG-1 gene and the biological behavior of colon cancer cells. It showed the remarkable advantage of RNAi in the generation of posttranscriptional gene silencing.
Insights
Researchers constructed shRNA plasmids targeting the human BAG-1 gene, successfully inhibiting its expression in colon cancer cells. This RNA interference (RNAi) method offers long-term gene silencing for cancer research.
Area of Science:
- Molecular Biology
- Cancer Research
- Gene Silencing
Background:
- The BAG-1 gene plays a role in cellular biological behavior.
- Understanding BAG-1's function is crucial for colon cancer research.
- RNA interference (RNAi) offers a method for targeted gene expression modulation.
Purpose of the Study:
- To construct short hairpin RNA (shRNA) plasmids targeting the human BAG-1 gene.
- To evaluate the efficacy of these shRNA plasmids in inhibiting BAG-1 gene expression at both mRNA and protein levels.
- To establish a stable cell line for long-term study of BAG-1 gene function in colon cancer.
Main Methods:
- Gene cloning techniques were used to construct recombinant plasmids with green fluorescent protein (GFP) reporter genes.
- RNA interference (RNAi) technology was employed to create shRNA plasmids specific to the BAG-1 gene.
- RT-PCR and Western blot assays were utilized to assess gene and protein expression levels post-transfection in Lo Vo colon cancer cells.
Main Results:
- Successfully constructed and recombined specific shRNA plasmids for the human BAG-1 gene.
- Established stably transfected Lo Vo colon cancer cell lines.
- Demonstrated significant and long-term inhibition of both BAG-1 mRNA and protein expression in transfected cells.
Conclusions:
- The developed shRNA plasmids effectively inhibit BAG-1 gene expression in colon cancer cells.
- The sequence pGPH1/GFP/Neo-BAG-1-homo-825 was identified as the optimal interference sequence.
- RNAi technology provides a powerful tool for post-transcriptional gene silencing in cancer research.
