A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans
A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans
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DOI：
10.1093/nar/28.22.e97
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发表时间：
2000-11-15
影响因子：
14.9
通讯作者：
d'Enfert, Christophe
中科院分区：
文献类型：
--
作者：
Chaveroche, Marie-Kim;Ghigo, Jean-Marc;d'Enfert, Christophe
突变体真菌菌株的构建通常受到这些生物体中同源重组效率低的限制。通过增加转化标记侧翼的同源DNA的长度可以获得更高的重组效率，尽管当使用标准分子生物学技术构建基因置换盒时，这是一个冗长的过程。在这里，我们提出了一个两步技术，利用大肠杆菌菌株表达噬菌体λ红（gam，bet，exo）起作用，并涉及（i）在该菌株中通过携带感兴趣的基因组区域的粘粒与PCR产生的转化标记之间的体内重组来构建重组粘粒，所述转化标记侧翼为与靶DNA同源的50 bp区域，和（ii）真菌自身中染色体基因座与环状或线性重组粘粒之间的遗传交换。该策略使得能够以> 50%的效率快速建立携带基因敲除的突变菌株。它也应该适合于构建具有基因融合或启动子置换的真菌菌株。
The construction of mutant fungal strains is often limited by the poor efficiency of homologous recombination in these organisms. Higher recombination efficiencies can be obtained by increasing the length of homologous DNA flanking the transformation marker, although this is a tedious process when standard molecular biology techniques are used for the construction of gene replacement cassettes. Here, we present a two-step technology which takes advantage of an Escherichia coli strain expressing the phage lambda Red(gam, bet, exo) functions and involves (i) the construction in this strain of a recombinant cosmid by in vivo recombination between a cosmid carrying a genomic region of interest and a PCR-generated transformation marker flanked by 50 bp regions of homology with the target DNA and (ii) genetic exchange in the fungus itself between the chromosomal locus and the circular or linearized recombinant cosmid. This strategy enables the rapid establishment of mutant strains carrying gene knock-outs with efficiencies >50%. It should also be appropriate for the construction of fungal strains with gene fusions or promoter replacements.