Scarless and site-directed mutagenesis in Salmonella enteritidis chromosome.
Scarless and site-directed mutagenesis in Salmonella enteritidis chromosome.
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DOI：
10.1186/1472-6750-7-59
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发表时间：
2007-09-17
影响因子：
3.5
通讯作者：
Kwon YM
中科院分区：
文献类型：
--
作者：
Cox MM;Layton SL;Jiang T;Cole K;Hargis BM;Berghman LR;Bottje WG;Kwon YM
已经描述了各种技术，其引入无瘢痕的位点特异性染色体突变。这些技术可应用于点突变或基因缺失以及将异源DNA插入细菌载体用于疫苗开发。大多数方法使用需要克隆和/或设计重复序列以促进同源重组的多步骤方法。我们已经修改了以前发表的技术，以开发一种简单，有效的PCR为基础的方法，无瘢痕插入到沙门氏菌染色体的DNA。这种突变策略的最终产物是将编码外源表位的DNA插入S。在不添加任何不需要的序列的情况下扩增基因组。该实验通过两步突变过程进行，所述两步突变过程经由PCR片段、Red重组酶和使用I-SceI酶位点的反选择。首先，将I-SceI位点和卡那霉素抗性基因导入表达Red重组酶的细胞的基因组中。接下来，用选择的插入序列替换该序列。用于重组的DNA片段是线性PCR产物，其由侧翼为靶基因的同源序列的外源插入序列组成。本文描述了将甲型流感病毒的M2 e表位（LM 2）的一部分、CD 154的结构域（CD 154 s）或两者的组合插入S的外膜蛋白LamB中。好吧我们已经成功地使用这种方法产生了多个突变体，除了表位区域表达所需的那些核苷酸外，基因组上没有抗生素基因或额外序列。该方法优于其他方案，因为它不需要克隆或产生额外的重复区域以促进同源重组，含有通用构建体，其中可以放置选择的表位以检查细胞表面表达，并且在筛选阳性突变体时显示出高效率。这种突变策略的其他机会包括产生减毒突变体和位点特异性染色体缺失突变。此外，该方法应该适用于Red重组酶可以功能性表达的其他革兰氏阴性细菌物种。
A variety of techniques have been described which introduce scarless, site-specific chromosomal mutations. These techniques can be applied to make point mutations or gene deletions as well as insert heterologous DNA into bacterial vectors for vaccine development. Most methods use a multi-step approach that requires cloning and/or designing repeat sequences to facilitate homologous recombination. We have modified previously published techniques to develop a simple, efficient PCR-based method for scarless insertion of DNA into Salmonella enteritidis chromosome. The final product of this mutation strategy is the insertion of DNA encoding a foreign epitope into the S. enteritidis genome without the addition of any unwanted sequence. This experiment was performed by a two-step mutation process via PCR fragments, Red recombinase and counter-selection with the I-SceI enzyme site. First, the I-SceI site and kanamycin resistance gene were introduced into the genome of cells expressing Red recombinase enzymes. Next, this sequence was replaced by a chosen insertion sequence. DNA fragments used for recombination were linear PCR products which consisted of the foreign insertion sequence flanked by homologous sequences of the target gene. Described herein is the insertion of a section of the M2e epitope (LM2) of Influenza A virus, a domain of CD154 (CD154s) or a combination of both into the outer membrane protein LamB of S. enteritidis. We have successfully used this method to produce multiple mutants with no antibiotic gene on the genome or extra sequence except those nucleotides required for expression of epitope regions. This method is advantageous over other protocols in that it does not require cloning or creating extra duplicate regions to facilitate homologous recombination, contains a universal construct in which an epitope of choice can be placed to check for cell surface expression, and shows high efficiency when screening for positive mutants. Other opportunities of this mutational strategy include creating attenuated mutants and site-specific, chromosomal deletion mutations. Furthermore, this method should be applicable in other gram-negative bacterial species where Red recombinase enzymes can be functionally expressed.
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影响因子：
4.4
作者：
Liu, WL;Zou, P;Chen, YH
通讯作者：
Chen, YH
DOI：
10.1073/pnas.120163297
发表时间：
2000-06-06
影响因子：
11.1
作者：
Datsenko, KA;Wanner, BL
通讯作者：
Wanner, BL
影响因子：
56.9
作者：
Rüssmann, H;Shams, H;Donis, RO
通讯作者：
Donis, RO
影响因子：
6.4
作者：
Vega, MI;Santos-Argumedo, L;González-Bonilla, CR
通讯作者：
González-Bonilla, CR
影响因子：
3.1
作者：
Lu, SW;Killoran, PB;Riley, LW
通讯作者：
Riley, LW