Development of a highly efficient gene targeting system induced by transient repression of YKU80 expression in Candida glabrata
Development of a highly efficient gene targeting system induced by transient repression of YKU80 expression in Candida glabrata
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DOI：
10.1128/ec.00414-06
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发表时间：
2007-07-01
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影响因子：
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通讯作者：
Chibana, Hiroji
中科院分区：
文献类型：
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作者：
Ueno, Keigo;Uno, Jun;Chibana, Hiroji
在致病性酵母光滑假丝酵母中，基因靶向产生敲除和“敲入”是分析基因功能的潜在有力方法。在光滑念珠菌基因组测序计划之后，其重要性增加，但该领域的进展受到基因靶向机制效率低下的阻碍。使用40-bp同源侧翼DNA，未鉴定到基因靶向。为了解决这个问题，YKU 80被破坏，导致使用40-bp侧翼同源DNA的靶向效率增加5.1%。为了利用YKU 80失活对基因靶向频率的有益影响而不引起任何负面影响，如合成病或致死性，我们开发了一种新的系统，其中在转化期间瞬时敲低表达后恢复YKU 80的表达。用于该新系统的菌株在YKU 80启动子区域中携带SAT 1翻转器，该翻转器用于在转化期间抑制表达，但在转化后自发地从基因座切除。通过使用该菌株，甲基甲烷磺酸甲酯，112021紫外线照射，和羟基脲诱导的DNA损伤之前和基因打靶过程中进行了评估和URA 3的突变率测定。没有显着的影响，SAT 1鳍对这些进程已被确定。在切除SAT 1翻转器后，在启动子区域中留下34-bp FLP重组靶序列。然而，mRNA转录水平得到恢复，并且与YKU 80野生型菌株相比，体内存活率没有差异。
In the pathogenic yeast Candida glabrata, gene targeting to generate knockouts and "knockins" is a potentially powerful method for the analysis of gene function. Its importance increased after the C glabrata genome sequence project, but progress in the field is hampered by inefficient mechanisms for gene targeting. With the use of 40-bp homologous flanking DNA, no gene targeting was identified. To address this issue, YKU80 was disrupted, leading to an increase in targeting efficiency of 5.1% using 40-bp flanking homologous DNA. To harness the beneficial effects of YKU80 inactivation on gene targeting frequency without incurring any negative effects, such as synthetic sickness or lethality, we developed a new system whereby the expression of YKU80 was restored following a transient knockdown of expression during transformation. Strains used for this new system carried a SAT1 flipper in the YKU80 promoter region, which was used to repress expression during transformation but was spontaneously excised from the locus after the transformation. By using this strain, DNA damage induced by methyl methane sulfonate, 112021 UV irradiation, and hydroxyurea before and during gene targeting was evaluated and the mutation rate of URA3 was determined. No significant effects of the SAT1 flipper on these processes have been identified. After the SAT1 flipper is excised, a 34-bp FLP recombination target sequence is left in the promoter region. However, the levels of mRNA transcription were restored and no difference in the survival ratio in vivo compared to that with the YKU80 wild-type strain was identified.