GENE TARGETS OF ANTIMICROSPORIDIAL DRUGS

抗微孢子虫药物的基因靶点

基本信息

批准号：
7958587
负责人：
Elizabeth Schmidt Didier
金额：
$ 6.05万
依托单位：
TULANE UNIVERSITY OF LOUISIANA
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-05-01 至 2010-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7958587
关键词：
Address Biological Assay Buffers Cells Computer Retrieval of Information on Scientific Projects Database Confocal Microscopy Cornea Diarrhea Double-Stranded RNA Electroporation Extracellular Matrix Fluorescein Fluoresceins Funding Gene Targeting Genes Glycerol Grant Human Infection Institution Label Measures Methods Microsporidia Nosema corneum Oligonucleotides Organism Pharmaceutical Preparations Physiologic pulse Primates RNA Interference Reproduction spores Research Research Personnel Resources Source Staging Sucrose Systemic disease Techniques Transfection United States National Institutes of Health gene function genetic manipulation invertebrate host member uptake

Address Biological Assay Buffers Cells Computer Retrieval of Information on Scientific Projects Database Confocal Microscopy Cornea Diarrhea Double-Stranded RNA Electroporation Extracellular Matrix Fluorescein Fluoresceins Funding Gene Targeting Genes Glycerol Grant Human Infection Institution Label Measures Methods Microsporidia Nosema corneum Oligonucleotides Organism Pharmaceutical Preparations Physiologic pulse Primates RNA Interference Reproduction spores Research Research Personnel Resources Source Staging Sucrose Systemic disease Techniques Transfection United States National Institutes of Health gene function genetic manipulation invertebrate host member uptake

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项目摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Members of the phylum Microsporidia are single-celled obligate intracellular protists that infect vertebrate and invertebrate hosts. Fourteen species of microsporidia have been identified as causes of opportunistic and emerging infections in humans and have been associated with persistent diarrhea and systemic disease. Studies on gene function in the microsporidia have been limited by the lack of genetic manipulation techniques, in part due to the presence of a chitinous wall that surrounds the mature infectious spore stage. To begin to address this issue, electroporation methods were employed to determine if a fluorescein-labeled dsRNA oligonucleotide (BLOCK-iT; Invitrogen) could be introduced into the spores of the human microsporidian species, Vittaforma corneae. Organisms were suspended in various electroporation buffers and subjected to multiple exponential decay and square wave electric pulses in the presence of the FITC-dsRNA. The conditions that resulted in optimal uptake of the FITC-dsRNA, as assessed by confocal microscopy, were electroporation of V. corneae in a 4 mm gap cuvette suspended in electroporation buffer (5 mM CaCl2, 10 mM NaCl, 8.7% glycerol, 0.2 M sucrose, 10 mM HEPES, pH 6.8) and exposed to 2 pulses of 2kV/cm for 80 ¿sec each using the ECM 830 electroporation unit (BTX). Efficiency of the FITC-oligonucleotide transfection was 19.54% (+ 1.05) and viability, as measured by a limiting-dilution infectivity assay, was reduced by approximately 50%. These results require further optimization to increase transfection efficiency, but open the door to the application of electroporation for studies on gene function and gene manipulation by, for example, RNA interference, in the microsporidia.

该副本是使用众多研究子项目之一由NIH/NCRR资助的中心赠款提供的资源。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构是对于中心，这是调查员的机构。门孢子虫的成员是感染脊椎动物和无脊椎动物宿主的单细胞内细胞内生物。已确定有14种微孢子虫是人类的机会性和新兴感染的原因，并且与持续性腹泻和全身性疾病有关。缺乏遗传操纵技术，有关微孢子虫中基因功能的研究受到限制，部分原因是存在围绕成熟感染性酱汁阶段的几点壁壁。为了开始解决这个问题，采用电穿孔方法来确定是否可以将荧光素标记的dsRNA寡核苷酸（块状IT; Invitrogen）引入人类微孢子虫物种Vittaforma Corneae。在FITC-DSRNA存在下，将生物体悬浮在各种电穿孔缓冲液中，并经历多种指数衰减和方波电脉冲。 The conditions that resulted in optimal uptake of the FITC-dsRNA, as assessed by confocal microscopy, were electroporation of V. corneae in a 4 mm gap cuvette suspended in electroporation buffer (5 mM CaCl2, 10 mM NaCl, 8.7% glycerol, 0.2 M sucrose, 10 mM HEPES, pH 6.8) and exposed to 2 pulses of 2kV/cm for 80 �使用ECM 830电穿孔单元（BTX）sec。 FITC-寡核苷酸转化的效率为19.54％（+ 1.05），通过限制稀释感染评估来衡量的生存力降低了约50％。这些结果需要进一步的优化以提高转化效率，但在微孢子虫中，通过RNA干扰将电穿孔应用于基因功能和基因操纵的研究。