Optimization of feline immunodeficiency virus vectors for RNA interference
Optimization of feline immunodeficiency virus vectors for RNA interference
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DOI：
10.1128/jvi.00958-06
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发表时间：
2006-10-01
影响因子：
5.4
通讯作者：
Davidson, Beverly L.
中科院分区：
文献类型：
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作者：
Harper, Scott Q.;Staber, Patrick D.;Davidson, Beverly L.
RNA干扰(RNAi)作为一种调节基因表达的手段，在植物和动物细胞中自然存在。这一过程已经被实验操纵，以实现细胞、组织和动物中的靶向基因沉默，使用各种载体系统。在这里，我们测试了基于猫免疫缺陷病毒(FIV)的载体可以用于报告构建体和RNAi表达盒共表达的假设。出人意料的是，在我们最初的构建中，我们发现在聚合酶IOTA(PolIOTA IOTA)表达的报告基因下游放置RNAi表达盒抑制了报告基因的表达，但不抑制载体滴度。通过一系列中间载体的构建，我们发现RNAi表达盒相对于REV反应元件和PolIOTA表达盒的位置对于RNAi和报告基因的有效表达是至关重要的。这些结果表明，立体因素，包括RNA结构和竞争转录机制的招募，可能会影响来自FIV载体的基因表达。在第二系列研究中，我们发现在共表达报告基因的FIV载体和3‘非翻译区驻留的microRNAs转导的细胞中可以实现靶序列沉默。优化的基于FIV的RNAi表达载体将得到广泛的应用，因为假型FIV载体在体外和体内对多种细胞类型具有广泛的趋向性。
RNA interference (RNAi) occurs naturally in plant and animal cells as a means for modulating gene expression. This process has been experimentally manipulated to achieve targeted gene silencing in cells, tissues, and animals, using a variety of vector systems. Here, we tested the hypothesis that vectors based on feline immunodeficiency virus (FIV) could be used for coexpression of reporter constructs and RNAi expression cassettes. We found, unexpectedly, in our initial constructs that placement of RNAi expression cassettes downstream from a polymerase Iota Iota (pol Iota Iota)-expressed reporter gene inhibited reporter expression but not vector titer. Through a series of intermediate vector constructs, we found that placement of the RNAi expression cassette relative to the Rev response element and the pol Iota Iota expression cassette was critical for efficient RNAi and reporter gene expression. These results suggested that steric factors, including RNA structure and recruitment of competing transcriptional machinery, may affect gene expression from FIV vectors. In a second series of studies, we show that target sequence silencing can be achieved in cells transduced by FIV vectors coexpressing reporter genes and 3 ' untranslated region resident microRNAs. The optimized FIV-based RNAi expression vectors will find broad use given the extensive tropism of pseudotyped FIV vectors for many cell types in vitro and in vivo.