Targeted mutagenesis of dengue virus type 2 replicon RNA by yeast in vivo recombination.
Targeted mutagenesis of dengue virus type 2 replicon RNA by yeast in vivo recombination.
复制标题
通过酵母体内重组对登革热病毒 2 型复制子 RNA 进行定向诱变。
DOI：
10.1007/978-1-4939-0348-1_10
复制
发表时间：
2014
期刊：
影响因子：
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通讯作者：
Padmanabhan,Radhakrishnan
中科院分区：
文献类型：
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作者：
Manzano,Mark;Padmanabhan,Radhakrishnan
在黄病毒生物学研究中，利用cDNA感染性克隆或亚基因组复制子是必不可少的。突变的核苷酸或氨基酸残基为它们在病毒生命周期中的功能提供了重要线索。然而，建立黄病毒反向遗传系统的一个主要挑战是它们在大肠杆菌中的核苷酸序列不稳定。因此，使用传统的基于限制性内切酶的程序直接克隆通常会导致不必要的结构重排。在本章中，我们将讨论绕过传统克隆程序的克隆策略。我们利用以前研究中的观察结果，(1)细菌中的不稳定序列可以在真核系统中克隆，(2)酿酒酵母拥有一套研究得很好的遗传学系统，可以使用同源重组来引入序列。我们描述了一种在亚基因组登革病毒2复制子中进行靶向突变的方案。我们的方法利用酵母中的同源重组，使用线性化的复制子和包含所需突变的PCR产物。从该方法派生的构造可以被传播。大肠杆菌具有更好的稳定性。因此，酵母体内重组为黄病毒感染性克隆或复制子的基因工程提供了一种很好的策略，因为该系统与黄病毒固有的不稳定序列相容，不受传统克隆程序的限制。
The use of cDNA infectious clones or subgenomic replicons is indispensable in studying flavivirus biology. Mutating nucleotides or amino acid residues gives important clues to their function in the viral life cycle. However, a major challenge to the establishment of a reverse genetics system for flaviviruses is the instability of their nucleotide sequences inEscherichia coli. Thus, direct cloning using conventional restriction enzyme-based procedures usually leads to unwanted rearrangements of the construct. In this chapter, we discuss a cloning strategy that bypasses traditional cloning procedures. We take advantage of the observations from previous studies that (1) unstable sequences in bacteria can be cloned in eukaryotic systems and (2)Saccharomyces cerevisiaehas a well-studied genetics system to introduce sequences using homologous recombination. We describe a protocol to perform targeted mutagenesis in a subgenomic dengue virus 2 replicon. Our method makes use of homologous recombination in yeast using a linearized replicon and a PCR product containing the desired mutation. Constructs derived from this method can be propagated inE. coliwith improved stability. Thus, yeast in vivo recombination provides an excellent strategy to genetically engineer flavivirus infectious clones or replicons because this system is compatible with inherently unstable sequences of flaviviruses and is not restricted by the limitations of traditional cloning procedures.