New plasmid vectors for specific gene targeting in Spiroplasma citri
New plasmid vectors for specific gene targeting in Spiroplasma citri
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DOI：
10.1016/s0147-619x(02)00121-x
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发表时间：
2002-09-01
期刊：
影响因子：
2.6
通讯作者：
Renaudin, J
中科院分区：
文献类型：
--
作者：
Lartigue, C;Duret, S;Renaudin, J
在柑橘螺原体中，通过同源重组已经通过复制实现了基因失活。 oriC 质粒 pBOT1 作为破坏载体。然而。质粒重组需要转化体的广泛传代。在大多数情况下，重组发生在oriC而不是靶基因处。在当前的研究中，我们描述了一种新的载体，其中oriC片段被减少到能够促进质粒复制的最小序列。使用该载体使运动基因 scm1 失活表明 oriC 片段大小的减小确实增加了靶基因的重组频率。此外。为了避免转化体的广泛传代，我们开发了一种策略，其中只有当质粒通过靶基因处的一次交换重组整合到染色体中时，选择性的四环素抗性表型才能表达。作为一个例子，描述了螺旋蛋白基因的靶向。 (C) 2002 年爱思唯尔科学（美国）。版权所有。
In Spiroplasma citri gene inactivation through homologous recombination has been achieved by using the replicative. oriC plasmid pBOT1 as the disruption vector. However. plasmid recombination required extensive passaging of the transformants and. in most cases, recombination occurred at oriC rather than at the target gene. In the current study, we describe a new vector, in which the oriC fragment was reduced to the minimal sequences able to promote plasmid replication. Using this vector to inactivate the motility gene scm1 showed that size reduction of the oriC fragment did increase the frequency of recombination at the target gene. Furthermore. to avoid extensive passaging of the transformants, we developed a strategy in which the selective, tetracycline resistance phenotype can only be expressed once the plasmid has integrated into the chromosome by one single crossover recombination at the target gene. As an example, targeting of the spiralin gene is described. (C) 2002 Elsevier Science (USA). All rights reserved.