Seong Kyun Park1, Byung Joon Hwang1, Yun Kee2. 1. Department of Molecular Bioscience, College of Biomedical Science, Kangwon National University, Chuncheon, Kangwon-do, Republic of Korea. 2. Division of Biomedical Convergence, College of Biomedical Science, Kangwon National University, Chuncheon, Kangwon-do, Republic of Korea. yunkee@kangwon.ac.kr.
Abstract
BACKGROUND: RNA interference (RNAi), defined as double-stranded, RNA-mediated gene silencing, is a useful tool for functional genomic studies. Along with increasing information about genomic sequences due to the innovative development of genome-sequencing technologies, functional genomic technologies are needed to annotate the genome and determine the processes by which each gene is regulated. Lentiviral vectors have been used to efficiently deliver reagents, such as small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs), into cells and tissues for functional genomic analyses. OBJECTIVE: We developed a lentiviral vector that efficiently expresses intronic shRNA from the tetracycline regulatory element (TRE) promoter in a doxycycline-dependent manner. METHODS: We developed a lentiviral vector system that contains reverse tetracycline-controlled transactivator 3 (rtTA3) and the TRE promoter, which are necessary for the doxycycline-inducible expression of shRNAs that are expressed as intronic miR-30a precursors. We then measured the cross-talk between the cytomegalovirus (CMV) and TRE promoters in the vector. RESULTS: We found that nearby promoters influence each other and that the TRE promoter should be located far from other promoters, such as the CMV promoter, in a vector. The orientation of a promoter with respect to other promoters also influences its transcriptional activity. A head-to-head orientation of the CMV and TRE promoters maintains the lowest level of transcription from TRE in the absence of doxycycline, compared to the tail-to-tail and head-to-tail orientations. CONCLUSION: Based on these findings, we were able to construct a lentiviral vector that faithfully expresses intronic miR-30a shRNA precursors in a doxycycline-inducible manner.
BACKGROUND: RNA interference (RNAi), defined as double-stranded, RNA-mediated gene silencing, is a useful tool for functional genomic studies. Along with increasing information about genomic sequences due to the innovative development of genome-sequencing technologies, functional genomic technologies are needed to annotate the genome and determine the processes by which each gene is regulated. Lentiviral vectors have been used to efficiently deliver reagents, such as small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs), into cells and tissues for functional genomic analyses. OBJECTIVE: We developed a lentiviral vector that efficiently expresses intronic shRNA from the tetracycline regulatory element (TRE) promoter in a doxycycline-dependent manner. METHODS: We developed a lentiviral vector system that contains reverse tetracycline-controlled transactivator 3 (rtTA3) and the TRE promoter, which are necessary for the doxycycline-inducible expression of shRNAs that are expressed as intronic miR-30a precursors. We then measured the cross-talk between the cytomegalovirus (CMV) and TRE promoters in the vector. RESULTS: We found that nearby promoters influence each other and that the TRE promoter should be located far from other promoters, such as the CMV promoter, in a vector. The orientation of a promoter with respect to other promoters also influences its transcriptional activity. A head-to-head orientation of the CMV and TRE promoters maintains the lowest level of transcription from TRE in the absence of doxycycline, compared to the tail-to-tail and head-to-tail orientations. CONCLUSION: Based on these findings, we were able to construct a lentiviral vector that faithfully expresses intronic miR-30a shRNA precursors in a doxycycline-inducible manner.
Authors: Stéphane Palfi; Jean Marc Gurruchaga; G Scott Ralph; Helene Lepetit; Sonia Lavisse; Philip C Buttery; Colin Watts; James Miskin; Michelle Kelleher; Sarah Deeley; Hirokazu Iwamuro; Jean Pascal Lefaucheur; Claire Thiriez; Gilles Fenelon; Cherry Lucas; Pierre Brugières; Inanna Gabriel; Kou Abhay; Xavier Drouot; Naoki Tani; Aurelie Kas; Bijan Ghaleh; Philippe Le Corvoisier; Patrice Dolphin; David P Breen; Sarah Mason; Natalie Valle Guzman; Nicholas D Mazarakis; Pippa A Radcliffe; Richard Harrop; Susan M Kingsman; Olivier Rascol; Stuart Naylor; Roger A Barker; Philippe Hantraye; Philippe Remy; Pierre Cesaro; Kyriacos A Mitrophanous Journal: Lancet Date: 2014-01-10 Impact factor: 79.321
Authors: Dmitri Wiederschain; Susan Wee; Lin Chen; Alice Loo; Guizhi Yang; Alan Huang; Yan Chen; Giordano Caponigro; Yung-Mae Yao; Christoph Lengauer; William R Sellers; John D Benson Journal: Cell Cycle Date: 2009-02-25 Impact factor: 4.534