Silencing Genes in the Heart
Silencing Genes in the Heart
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DOI：
10.1007/978-1-4939-6588-5_2
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发表时间：
2017-01-01
期刊：
影响因子：
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通讯作者：
Poller, Wolfgang
中科院分区：
文献类型：
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作者：
Fechner, Henry;Vetter, Roland;Poller, Wolfgang
利用RNA干扰（RNAi）技术沉默心脏基因已成为治疗心脏疾病的一种新方法。小干扰RNA是RNAi的诱导物，但培养的原代心肌细胞和心脏对siRNA转染具有高度抗性。这可以通过由亲心腺相关病毒（AAV）载体递送小发夹（sh）RNA或人工微小RNA（amiRNA）来克服。在这里，我们描述了作为心脏基因沉默的例子，针对心脏蛋白受磷蛋白的shRNA和amiRNA的产生和克隆。我们进一步描述了具有自身互补载体基因组的AAV穿梭质粒的产生、在滚瓶中的AAV载体的生产以及它们通过碘克沙醇梯度离心和用过滤系统浓缩的纯化。最后，我们描述了原代新生大鼠心肌细胞（PNRC）的制备，用AAV载体转导PNRC，以及转导细胞培养物的维持。
Silencing of cardiac genes by RNA interference (RNAi) has developed into a powerful new method to treat cardiac diseases. Small interfering (si) RNAs are the inducers of RNAi, but cultured primary cardiomyocytes and heart are highly resistant to siRNA transfection. This can be overcome by delivery of small hairpin (sh)RNAs or artificial microRNA (amiRNAs) by cardiotropic adeno-associated virus (AAV) vectors. Here we describe as example of the silencing of a cardiac gene, the generation and cloning of shRNA, and amiRNAs directed against the cardiac protein phospholamban. We further describe the generation of AAV shuttle plasmids with self complementary vector genomes, the production of AAV vectors in roller bottles, and their purification via iodixanol gradient centrifugation and concentration with filter systems. Finally we describe the preparation of primary neonatal rat cardiomyocytes (PNRC), the transduction of PNRC with AAV vectors, and the maintenance of the transduced cell culture.