PDF(Pubmed)
Abstract：
In virology, the term reverse genetics refers to a set of methodologies in which changes are introduced into the viral genome and their effects on the generation of infectious viral progeny and their phenotypic features are assessed. Reverse genetics emerged thanks to advances in recombinant DNA technology, which made the isolation, cloning, and modification of genes through mutagenesis possible. Most virus reverse genetics studies depend on our capacity to rescue an infectious wild-type virus progeny from cell cultures transfected with an \"infectious clone\". This infectious clone generally consists of a circular DNA plasmid containing a functional copy of the full-length viral genome, under the control of an appropriate polymerase promoter. For most DNA viruses, reverse genetics systems are very straightforward since DNA virus genomes are relatively easy to handle and modify and are also (with few notable exceptions) infectious per se. This is not true for RNA viruses, whose genomes need to be reverse-transcribed into cDNA before any modification can be performed. Establishing reverse genetics systems for members of the Caliciviridae has proven exceptionally challenging due to the low number of members of this family that propagate in cell culture. Despite the early successful rescue of calicivirus from a genome-length cDNA more than two decades ago, reverse genetics methods are not routine procedures that can be easily extrapolated to other members of the family. Reports of calicivirus reverse genetics systems have been few and far between. In this review, we discuss the main pitfalls, failures, and delays behind the generation of several successful calicivirus infectious clones.
摘要：
在病毒学中,术语反向遗传学是指一组方法，其中将变化引入病毒基因组，并评估其对感染性病毒后代产生的影响及其表型特征。反向遗传学的出现得益于重组DNA技术的进步,这使得隔离，克隆,和通过诱变可能的基因修饰。大多数病毒反向遗传学研究依赖于我们从“感染性克隆”转染的细胞培养物中拯救感染性野生型病毒后代的能力。这种感染性克隆通常由含有全长病毒基因组的功能拷贝的环状DNA质粒组成。在适当的聚合酶启动子的控制下。对于大多数DNA病毒，反向遗传学系统非常简单，因为DNA病毒基因组相对容易处理和修饰，并且本身也具有传染性（很少有值得注意的例外）。RNA病毒并非如此，在进行任何修饰之前，其基因组需要逆转录为cDNA。由于该家族在细胞培养物中繁殖的成员数量少，因此为杯状病毒科成员建立反向遗传学系统已被证明是非常具有挑战性的。尽管二十多年前从基因组长度的cDNA中早期成功地拯救了杯状病毒，反向遗传学方法不是常规程序，可以很容易地推断到家庭的其他成员。关于杯状病毒反向遗传学系统的报道很少。在这次审查中,我们讨论主要的陷阱，失败,并延迟了几个成功的杯状病毒感染性克隆的产生。
