Hung-Yao Ho1, Mei-Ling Cheng, Yi-Hui Wang, Daniel Tsun-Yee Chiu. 1. Graduate Institute of Medical Biotechnology and Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Kwei-san, Tao-yuan, Taiwan.
Abstract
BACKGROUND: Small interfering RNA (siRNA) has emerged as a powerful tool to study the loss-of-function phenotype by specifically silencing a target gene. The success of gene silencing depends on the choice of appropriate target sequence, and requires a rapid and sensitive assay for quantifying siRNA efficiency. Conventional assays include Western blotting and reverse transcription coupled with polymerase chain reaction. However, these methods are somewhat inaccurate owing to the variation in transfection efficiency. To avoid this, we have developed a flow cytometric method to provide a quantitative analysis of siRNA efficacy. METHODS: We constructed a novel vector pHyper1G, which can express enhanced green fluorescent protein (EGFP). It contains a H1 promoter, which can drive expression of short hairpin RNA (shRNA) directed against a target gene. The target gene was cloned into pDsRed1-N1. The resulting construct can express a fusion protein between target protein and DsRed. These vectors were co-transfected into 293T cells. The transfected cells were analyzed by flow cytometry. The percentage of EGFP+, DsRed+ cells and the change in mean fluorescence intensity (MFI) of DsRed channel indicate changes in expression of target gene in a cell population, and hence the efficacy of the corresponding shRNA. In addition, the cells transfected with pHyper1G derivative were sorted, and analyzed for the activity of target gene. RESULTS: We designed an oligonucleotide duplex encoding shRNA against glucose-6-phosphate dehydrogenase (G6PD) gene, and cloned this into pHyper1G. The resulting vector pHyper1G101 effectively knocked down the expression of G6PD-DsRed from pDsRed301, as shown by significant reduction in both the percentage of EGFP+, DsRed+ cells, and MFI. Changes in these parameters were consistent with decreases in protein level and activity of G6PD. Moreover, albeit at low suboptimal transfection efficiency, cells transfected with pHyper1G101 alone were successfully sorted for those expressing shRNA, and their G6PD activity was found to be suppressed. CONCLUSION: Flow cytometric analysis provides a reliable assessment of the efficiency of siRNA in a cell population. This method can be easily automated and used for screening of appropriate shRNAs against certain genes. Moreover, using the present system, we can deliberately sort for and analyze those cells expressing shRNA. This can be applied to the hard-to-transfect cell types, and greatly facilitates the analyses of gene silencing in these cells.
BACKGROUND: Small interfering RNA (siRNA) has emerged as a powerful tool to study the loss-of-function phenotype by specifically silencing a target gene. The success of gene silencing depends on the choice of appropriate target sequence, and requires a rapid and sensitive assay for quantifying siRNA efficiency. Conventional assays include Western blotting and reverse transcription coupled with polymerase chain reaction. However, these methods are somewhat inaccurate owing to the variation in transfection efficiency. To avoid this, we have developed a flow cytometric method to provide a quantitative analysis of siRNA efficacy. METHODS: We constructed a novel vector pHyper1G, which can express enhanced green fluorescent protein (EGFP). It contains a H1 promoter, which can drive expression of short hairpin RNA (shRNA) directed against a target gene. The target gene was cloned into pDsRed1-N1. The resulting construct can express a fusion protein between target protein and DsRed. These vectors were co-transfected into 293T cells. The transfected cells were analyzed by flow cytometry. The percentage of EGFP+, DsRed+ cells and the change in mean fluorescence intensity (MFI) of DsRed channel indicate changes in expression of target gene in a cell population, and hence the efficacy of the corresponding shRNA. In addition, the cells transfected with pHyper1G derivative were sorted, and analyzed for the activity of target gene. RESULTS: We designed an oligonucleotide duplex encoding shRNA against glucose-6-phosphate dehydrogenase (G6PD) gene, and cloned this into pHyper1G. The resulting vector pHyper1G101 effectively knocked down the expression of G6PD-DsRed from pDsRed301, as shown by significant reduction in both the percentage of EGFP+, DsRed+ cells, and MFI. Changes in these parameters were consistent with decreases in protein level and activity of G6PD. Moreover, albeit at low suboptimal transfection efficiency, cells transfected with pHyper1G101 alone were successfully sorted for those expressing shRNA, and their G6PD activity was found to be suppressed. CONCLUSION: Flow cytometric analysis provides a reliable assessment of the efficiency of siRNA in a cell population. This method can be easily automated and used for screening of appropriate shRNAs against certain genes. Moreover, using the present system, we can deliberately sort for and analyze those cells expressing shRNA. This can be applied to the hard-to-transfect cell types, and greatly facilitates the analyses of gene silencing in these cells.
Authors: Cyrielle Doigneaux; Anthony M Pedley; Ishna N Mistry; Monika Papayova; Stephen J Benkovic; Ali Tavassoli Journal: J Biol Chem Date: 2020-05-21 Impact factor: 5.157