使用合成的、密码子优化的Cre-loxP系统对白色念珠菌进行基因破坏。

Gene disruption in Candida albicans using a synthetic, codon-optimised Cre-loxP system.

作者信息

Dennison Paul M J, Ramsdale Mark, Manson Claire L, Brown Alistair J P

机构信息

Aberdeen Fungal Group, School of Medical Sciences, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK.

出版信息

Fungal Genet Biol. 2005 Sep;42(9):737-48. doi: 10.1016/j.fgb.2005.05.006.

DOI:10.1016/j.fgb.2005.05.006

PMID:16043373

Abstract

The development of the molecular toolbox for the fungal pathogen Candida albicans has been hampered by its lack of an exploitable sexual cycle, its diploid nature, and its non-canonical genetic code. We describe the adaptation of the Cre-loxP site-specific recombination system as a tool for the efficient and controlled disruption of C. albicans genes. We have validated this system by disrupting two C. albicans loci: ADE2 and MET15. Ade2 and met15 null mutants were made using loxP-flanked ARG4- and HIS1-based disruption cassettes. These markers were then resolved from the C. albicans genome using a synthetic codon-optimised cre recombinase gene, with near 100% efficiency. Finally, CIp plasmids containing the URA3, HIS1, and ARG4 markers were generated for the reintegration of markers and target genes in control strains. This system allows multiple and sequential genetic manipulations, which will facilitate the functional analysis of multigene families in C. albicans.

摘要

白色念珠菌这种真菌病原体分子工具箱的开发一直受到其缺乏可利用的有性生殖周期、其二倍体性质以及其非标准遗传密码的阻碍。我们描述了将Cre-loxP位点特异性重组系统改编为一种有效且可控地破坏白色念珠菌基因的工具。我们通过破坏两个白色念珠菌基因座：ADE2和MET15，验证了该系统。使用基于loxP侧翼的ARG4和HIS1的破坏盒构建了Ade2和met15缺失突变体。然后使用合成的密码子优化的cre重组酶基因，以接近100%的效率从白色念珠菌基因组中去除这些标记。最后，构建了含有URA3、HIS1和ARG4标记的CIp质粒，用于在对照菌株中重新整合标记和靶基因。该系统允许进行多次和连续的基因操作，这将有助于对白色念珠菌中多基因家族进行功能分析。