Department of Physiology, West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, Chengdu, Sichuan, China.
Department of Forensic Pathology, West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, Chengdu, Sichuan, China.
PeerJ. 2023 May 17;11:e15433. doi: 10.7717/peerj.15433. eCollection 2023.
The miniMOS technique has been widely used in the community to generate single copy insertions. A worm is considered as a potential insertion candidate if it is resistant to G418 antibiotics and does not express a co-injected fluorescence marker. If the expression of the extrachromosomal array is very low, it is possible for a worm to be mistakenly identified as a miniMOS candidate, as this low expression level can still confer resistance to G418 without producing a detectable fluorescence signal from the co-injection marker. This may increase the workload for identifying the insertion locus in the subsequent steps. In the present study, we modified the plasmid platform for miniMOS insertion by incorporating a promoter-driven TagRFP or a ubiquitous H2B::GFP expression cassette into the targeting vector and introducing two loxP sites flanking the selection cassettes. Based on this new miniMOS tool kit, the removable fluorescence reporters can be used to visualize the single copy insertions, greatly reducing insertion locus identification efforts. In our experience, this new platform greatly facilitates the isolation of the miniMOS mutants.
miniMOS 技术已在该领域被广泛用于产生单拷贝插入。如果一个虫体能耐受 G418 抗生素且不表达共注射的荧光标记物，那么它就被认为是潜在的插入候选物。如果染色体外阵列的表达水平非常低，那么该虫体有可能被错误地鉴定为 miniMOS 候选物，因为这种低表达水平仍然可以赋予 G418 抗性，而不会从共注射标记物中产生可检测的荧光信号。这可能会增加后续步骤中鉴定插入位点的工作量。在本研究中，我们通过将启动子驱动的 TagRFP 或泛在的 H2B::GFP 表达盒整合到靶向载体中，并在选择盒两侧引入两个 loxP 位点，对 miniMOS 插入的质粒平台进行了修改。基于这个新的 miniMOS 工具包，可以使用可移动的荧光报告基因来可视化单拷贝插入，大大减少了插入位点鉴定的工作。根据我们的经验，这个新平台极大地促进了 miniMOS 突变体的分离。
