Cryptococcus neoformans gene expression in minimal media of wild type (WT) and ALL2 mutant (all2Δ) cells. Cryptococcus neoformans

To examine the comparative transcription profile of WT and all2Δ mutant cells in in vitro conditions to determine genes responsible for hypervirulent phenotype of all2Δ cells. Using transcriptional profiling, we determined that many genes involved in transport were differentially transcribed between the wild type and the mutant strain. With the help of further experiments we could rule out that ALL2 has a unique function in maintaining the intracellular pH under acidic conditions. Overall design: For arrays, total RNA was isolated from WT and all2Δ mutant using Qiagen RNeasy Kit. For RNA extraction, WT and all2Δ mutant were grown in minimal media. Cells were grown for overnight, diluted 1:100 and then again grown overnight or up to an OD of 0.6-0.7 with agitation (150rpm) at 37°C.

本研究旨在比较体外培养条件下野生型（Wild Type，WT）与all2Δ突变体细胞的转录谱，以鉴定介导all2Δ突变体细胞高毒力表型的相关基因。通过转录组分析，我们发现野生型菌株与突变株间诸多参与转运过程的基因存在差异转录。后续实验进一步排除了ALL2在酸性环境下维持细胞内pH的独特功能。整体实验设计如下：针对基因芯片实验，采用Qiagen RNeasy试剂盒从WT与all2Δ突变体细胞中提取总RNA。用于RNA提取的细胞均在基础培养基中培养：先过夜培养，按1:100比例稀释后，于37℃、150rpm振荡培养过夜或至光密度（Optical Density，OD）值达到0.6-0.7。