Conditional gene deletion with DiCre demonstrates an essential role for CRK3 in Leishmania mexicana cell cycle regulation.
Conditional gene deletion with DiCre demonstrates an essential role for CRK3 in Leishmania mexicana cell cycle regulation.
复制标题
DOI：
10.1111/mmi.13375
复制
发表时间：
2016-06
影响因子：
3.6
通讯作者：
Mottram JC
中科院分区：
文献类型：
--
作者：
Duncan SM;Myburgh E;Philipon C;Brown E;Meissner M;Brewer J;Mottram JC
墨西哥利什曼原虫有一个大家族的细胞周期蛋白依赖性激酶（CDK），反映了细胞周期和生命周期进程之间的复杂相互作用。来自先前研究的证据表明，与细胞周期蛋白CYC 6复合的Cdc 2相关激酶3（CRK 3）是主要细胞周期调节因子CDK 1的功能同源物，但缺乏在寄生虫增殖中起重要作用的明确遗传证据。为了解决这个问题，我们已经实施了一个诱导型基因缺失系统的基础上的二聚Cre重组酶（diCre）的目标CRK 3和阐明其在细胞周期中的作用，L。墨西哥。用雷帕霉素诱导前鞭毛体中的diCre活性导致floxed CRK 3的有效缺失，导致G2/M生长停滞。CRK 3转基因在雷帕霉素诱导的CRK 3缺失期间的共表达导致生长的互补，而活性位点CRK 3 T178 E突变体的表达则没有，这表明蛋白激酶活性对CRK 3功能至关重要。CRK 3在稳定期前鞭毛体中的诱导性缺失导致小鼠的生长减弱，从而证实CRK 3作为有用的治疗靶点，diCre作为分析利什曼原虫必需基因的有价值的新工具。
Leishmania mexicana has a large family of cyclin‐dependent kinases (CDKs) that reflect the complex interplay between cell cycle and life cycle progression. Evidence from previous studies indicated that Cdc2‐related kinase 3 (CRK3) in complex with the cyclin CYC6 is a functional homologue of the major cell cycle regulator CDK1, yet definitive genetic evidence for an essential role in parasite proliferation is lacking. To address this, we have implemented an inducible gene deletion system based on a dimerised Cre recombinase (diCre) to target CRK3 and elucidate its role in the cell cycle of L. mexicana. Induction of diCre activity in promastigotes with rapamycin resulted in efficient deletion of floxed CRK3, resulting in G2/M growth arrest. Co‐expression of a CRK3 transgene during rapamycin‐induced deletion of CRK3 resulted in complementation of growth, whereas expression of an active site CRK3 T178E mutant did not, showing that protein kinase activity is crucial for CRK3 function. Inducible deletion of CRK3 in stationary phase promastigotes resulted in attenuated growth in mice, thereby confirming CRK3 as a useful therapeutic target and diCre as a valuable new tool for analyzing essential genes in Leishmania.