Delgado-Ramos Lidia, Marcos Ana T, Ramos-Guelfo María S, Sánchez-Barrionuevo Leyre, Smet Francis, Chávez Sebastián, Cánovas David
Departamento de Genética, Hospital Universitario Virgen del Rocío-CSIC-Universidad de Sevilla, Seville, Spain Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío-CSIC-Universidad de Sevilla, Seville, Spain.
Departamento de Genética, Hospital Universitario Virgen del Rocío-CSIC-Universidad de Sevilla, Seville, Spain.
G3 (Bethesda). 2014 Sep 19;4(11):2271-8. doi: 10.1534/g3.114.014357.
The analysis of filamentous fungi by flow cytometry has been impossible to date due to their filamentous nature and size. In this work, we have developed a method that combines single-spore microencapsulation and large-particle flow cytometry as a powerful alternative for the genetic analysis of filamentous fungi. Individual spores were embedded in monodisperse alginate microparticles and incubated in the appropriate conditions. Growth could be monitored by light or fluorescent microscopy and Complex Object Parametric Analyzer and Sorter large-particle flow cytometry. Microencapsulated Trichoderma and Aspergillus spores could germinate and grow inside the alginate capsules. Growth tests revealed that auxotrophic mutants required the appropriate nutrients and that pyrithiamine and glufosinate halted fungal growth of sensitive but not resistant strains. We used an Aspergillus nidulans, thermosensitive mutant in the cell-cycle regulator gene nimX(CDK1) as proof-of-concept to the detection and identification of genetic phenotypes. Sorting of the microparticles containing the clonal fungal mycelia proved the power of this method to perform positive and/or negative selection during genetic screenings.
由于丝状真菌的丝状性质和大小，迄今为止，通过流式细胞术对其进行分析是不可能的。在这项工作中，我们开发了一种方法，该方法将单孢子微囊化和大颗粒流式细胞术相结合，作为丝状真菌遗传分析的一种强大替代方法。将单个孢子包埋在单分散藻酸盐微粒中，并在适当条件下孵育。可以通过光学或荧光显微镜以及复杂物体参数分析仪和分选仪大颗粒流式细胞术监测生长情况。微囊化的木霉和曲霉孢子可以在藻酸盐胶囊内发芽并生长。生长试验表明，营养缺陷型突变体需要适当的营养物质，而硫胺素和草铵膦会阻止敏感但不耐药菌株的真菌生长。我们使用细胞周期调节基因nimX（CDK1）中的烟曲霉热敏突变体作为检测和鉴定遗传表型的概念验证。对含有克隆真菌菌丝体的微粒进行分选证明了该方法在遗传筛选过程中进行阳性和/或阴性选择的能力。
