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Regulation of u6 promoter Activity by Transcriptional Interference in Viral Vector-Based RNAi

Genomics, Proteomics & Bioinformatics 2010年第3期8卷 170-179页

作者： Linghu Nie Meghna Das Thakur Yumei Wang Qin Su Yongliang Zhao Yunfeng Feng Key Laboratory of Genome Sciences and Information Cancer Biology and Genetics Group Beijing Institute of Genomics Chinese Acadamy of Sciences Beijing 100029 China Departments of Medicine and Cell Biology Washington University St. Louis MO 63110 USA Immunocore Limited Abingdon Oxon OX14 4RX UK.

The direct negative impact of the transcriptional activity of one component on the second one in c/s is referred to as transcriptional interference （TI）. u6 is a type III RNA polymerase III promoter commonly used for driving small hairpin RNA （shRNA） expression in vector-based RNAi. In the design and construction of viral vectors, multiple transcription units may be arranged in close proximity in a space-limited vector. Determining if u6 promoter activity can be affected by TI is critical for the expression of target shRNA in gene therapy or loss-of-function studies. In this research, we designed and implemented a modified retroviral system where shRNA and exogenous gene expressions were driven by two independent transcriptional units. We arranged u6 promoter driving .shRNA expression and ubiC promoter in two promoter arrangements. In primary macrophages, we found u6 promoter activity was inhibited by ubiC promoter when in the divergent arrangement but not in tandem. In contrast, PKG promoter had no such negative impact. Instead of enhancing u6 promoter activity, CMV enhancer had significant negative impact on u6 promoter activity in the presence of ubiC promoter. Our results indicate that u6 promoter activity can be affected by TI in a proximal promoter-specific and arrange- ment-dependent manner.

关键词： transcriptional interference u6 promoter shRNA retrovirus

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Establishment of highly efficient transgenic system for black soldier fly(Hermetia illucens)

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Insect Science 2023年第4期30卷 888-900页

作者： Zongqing Kou Xingyu Luo Yuguo Jiang Bihui Chen Yu Song Yaohui Wang Jun Xui Jeffery K.Tomberlin Yongping Huang Key Laboratory of Insect Developmental and Evolutionary Biology Center for Excellence in Molecular Plant SciencesShanghai Insttute of Plant Physiology and EcologyChinese Academy of SciencesShanghaiChina university of Chinese Academy of Sciences BeijingChina Department of Entomology Texas A&M UniversityCollege StationTXUSA

The black soldier fly(BSF),Hermetia illucens,is a promising insect for miti-gating solid waste problems as its larvae are able to bioconvert organic waste into valuable *** recently reported a high-quality genome assembly of the BSF;analysis of this genome sequence will further the understanding of insect biology and identify genes that can be manipulated to improve efficiency of *** enable genetic manip-ulation of the BSF,we have established the first transgenic methods for this economically important *** cloned and identified the ubiquitous actin5C promoter(Hiactin5C-p3k)and 3 endogenous u6 promoters(Hiu6:1,Hiu6:2,and Hiu6:3).The Hiactin5C pro-moter was used to drive expression of a hyperactive variant of the piggyBac transposase,which exhibited up to 6-fold improvement in transformation rate when compared to the wild-type ***,we evaluated the 3 Hiu6 promoters using this trans-genic ***6:1 and Hiu6:2 promoters provided the highest knockdown efficiency with RNAi and are thus promising candidates for future Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)***,our findings provide valuable genetic engineering toolkits for basic research and genetic manipulation of the BSF.

关键词： actin5C promoter black soldier fly hyPBase transgenesis u6 promoter

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Establishment and utilization of a Tetracycline-controlled Inducible RNA Interfering System to Repress Gene Expression in Chronic Myelogenous Leukemia Cells

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Acta Biochimica et Biophysica Sinica 2005年第12期37卷 851-856页

作者： Fan YANG Yun ZHANG Ying-Li CAO Shu-Hui WANG Li LIu Department of Microbiology and Etiology,Institute of Basic Medical .sic Medical Sciences. Chinese Academy q/’Medical Sciences and Peking union Medical College.Beijing 100005.China DepartmentofMicrobiologyandEtiology InstituteofBasicMedicalSciencesChineseAcademyofMedicalSciencesandPekin

RNA interference (RNAi),a posttranscriptional gene silencing process mediated by small double-stranded RNA specifically complementary to the targeted transcript,has been used extensively in the developmentof novel therapeutic approaches against various human diseases including chronic myelogenous leukemia (CML).*** report the successful construction of a tetracycline-controlled siRNA in CML cell line K562.A K562cell line stably expressing the reverse tetracycline-controlled transactivator (rtTA) was constructed.A tetracy-cline responsive element(TRE) was integrated into the RNA polymerase Ⅲ promoter region of pBS/u6 thatwas used to drive specific siRNA to target the novel cytokine receptor-like factor 3 (CRLF3) gene,The resultsshow that rtTA was able to recognize the TRE to prevent siRNA-mediated exogenous and endogenous CRLF3gene ***,CRLF3-siRNA mediated gene repression could be induced in a dose-dependentmanner in the presence of ***,the inducible siRNAi system in K562 cells might be useful for thestudy of RNAi-mediated therapeutic approaches against CML.

关键词： inducible siRNA tetracycline reverse tetracycline-controlled transactivator u6 promoter CRLF3

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