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首页> 外文学位 >Inhibition of Apolipoprotein-L 11b (Apol11b) Gene Expression Using Small Interference RNA Technology During Erythroid Terminal Differentiation.
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Inhibition of Apolipoprotein-L 11b (Apol11b) Gene Expression Using Small Interference RNA Technology During Erythroid Terminal Differentiation.

机译：红细胞末端分化过程中使用小干扰RNA技术抑制载脂蛋白L 11b（Apol11b）基因表达。

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摘要

Erythroid Terminal Differentiation (ETD) is the process by which immature precursor cells differentiate and become mature erythrocytes. The process includes the synthesis and accumulation of hemoglobin, chromatin condensation, and extrusion of the nucleus. Additional alteration in lipid composition of erythrocyte membrane during this process may be influenced by a novel protein, Apol11b. The expression of this protein was to be disrupted using small interference RNA (siRNA) in this study. Murine Erythroleukemia (MEL) cells normally express Apol11b upon induction with dimethylsulfoxide and thus were used as a model for determining the level of silencing of the Apol11b gene using siRNA. Initial experiments were performed to optimize electroporation conditions using a green fluorescent protein (GFP) expressing plasmid, with GFP expression being used to assay success of electroporation. Inconsistent results from these experiments resulted in the application of chemical transfection and then finally lentiviral transduction methodologies. Replication-incompetent lentiviral particles with 3 different DNA constructs (GFP cDNA, Control shRNA and Apol11b shRNA) were successfully used to transduce the MEL cells and stable cell lines were generated. Stable clones containing GFP confirmed successful transduction through visible fluorescence under microscope. Western blotting with an antibody previously generated against Apol11b protein and quantitative Polymerase Chain Reaction (qPCR) were used to assess Apol11b knockdown. Though the western blotting results could not be interpreted, statistical analysis of qPCR experiments demonstrated effective silencing of the Apol11b gene in the MEL cells transduced with the Apol11b shRNA vs. nontransduced MEL cells.
机译：红系终末分化（ETD）是未成熟前体细胞分化并成为成熟红细胞的过程。该过程包括血红蛋白的合成和积累，染色质浓缩和核的挤出。在此过程中，红细胞膜脂质组成的其他变化可能会受到新蛋白Apol11b的影响。在这项研究中使用小干扰RNA（siRNA）破坏该蛋白的表达。鼠红细胞白血病（MEL）细胞通常在用二甲基亚砜诱导后表达Apol11b，因此被用作确定使用siRNA沉默Apol11b基因水平的模型。使用表达绿色荧光蛋白（GFP）的质粒进行了初步实验，以优化电穿孔条件，并使用GFP表达来测定电穿孔的成功率。这些实验的不一致结果导致应用了化学转染，然后最终应用了慢病毒转导方法。具有3种不同DNA构建体（GFP cDNA，Control shRNA和Apol11b shRNA）的无复制慢病毒颗粒已成功用于转导MEL细胞，并产生了稳定的细胞系。含有GFP的稳定克隆证实在显微镜下通过可见荧光成功转导。用先前产生的针对Apol11b蛋白的抗体进行的蛋白质印迹和定量聚合酶链反应（qPCR）用于评估Apol11b的敲低。尽管无法解释蛋白质印迹结果，但qPCR实验的统计分析表明，与未转导的MEL细胞相比，Apol11b shRNA转导的MEL细胞中Apol11b基因有效沉默。

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  • 作者

    Datta, Arani.;

  • 作者单位

    State University of New York at Buffalo.;

  • 授予单位 State University of New York at Buffalo.;
  • 学科 Biology Molecular.
  • 学位 M.S.
  • 年度 2011
  • 页码 159 p.
  • 总页数 159
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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