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首页> 外文期刊>Infection and immunity >Cryptococcus neoformans Differential Gene Expression Detected In Vitro and In Vivo with Green Fluorescent Protein
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Cryptococcus neoformans Differential Gene Expression Detected In Vitro and In Vivo with Green Fluorescent Protein

机译：新绿色隐球菌差异基因表达的体外和体内检测与绿色荧光蛋白。

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Synthetic green fluorescent protein (GFP) was used as a reporter to detect differential gene expression in the pathogenic fungusCryptococcus neoformans. Promoters from the C. neoformans actin, GAL7, or mating-type alpha pheromone (MFα1) genes were fused to GFP, and the resulting reporter genes were used to assess gene expression in serotype A C. neoformans. Yeast cells containing an integrated pACT::GFP construct demonstrated that the actin promoter was expressed during vegetative growth on yeast extract-peptone-dextrose medium. In contrast, yeast cells containing the inducible GAL7::GFP or MFα1::GFP reporter genes expressed significant GFP activity only during growth on galactose medium or V-8 agar, respectively. These findings demonstrated that the GAL7 and MFα1 promoters from a serotype D C. neoformans strain function when introduced into a serotype A strain. Because the MFα1 promoter is induced by nutrient deprivation and the MATα locus containing the MFα1 gene has been linked with virulence, yeast cells containing the pMFα1::GFP reporter gene were analyzed for GFP expression in the central nervous system (CNS) of immunosuppressed rabbits. In fact, significant GFP expression from the MFα1::GFP reporter gene was detected after the first week of a CNS infection. These findings suggest that there are temporal, host-specific cues that regulate gene expression during infection and that the MFα1 gene is induced during the proliferative stage of a CNS infection. In conclusion, GFP can be used as an effective and sensitive reporter to monitor specific C. neoformans gene expression in vitro, and GFP reporter constructs can be used as an approach to identify a novel gene(s) or to characterize known genes whose expression is regulated during infection.
机译：利用合成的绿色荧光蛋白（GFP）作为报告基因，检测病原真菌新隐球菌中的差异基因表达。 C的发起人。将新形成的肌动蛋白，GAL7或交配型α信息素（MFα1）基因与GFP融合，并将所得的报道基因用于评估A C血清型中的基因表达。新甲虫。包含整合的pACT :: GFP构建体的酵母细胞证明肌动蛋白启动子在酵母提取物-蛋白-葡萄糖培养基上的营养生长过程中表达。相反，仅在半乳糖培养基或V-8琼脂上生长的过程中，含有诱导型GAL7 :: GFP或MFα1:: GFP报告基因的酵母细胞才表达显着的GFP活性。这些发现表明，来自血清型D C的GAL7和MFα1启动子。新甲型菌株在引入A型​​血清型菌株时发挥功能。因为MFα1启动子是由营养剥夺诱导的，并且包含MFα1基因的MATα位点已经与毒力相关，所以分析了包含pMFα1:: GFP报告基因的酵母细胞在中枢神经系统中的GFP表达。 （CNS）免疫抑制的兔子。实际上，在CNS感染的第一周后，从MFα1:: GFP报告基因中检测到了明显的GFP表达。这些发现表明存在暂时的宿主特异性信号，可在感染过程中调节基因表达，并且MFα1基因是在CNS感染的增殖期诱导的。总之，GFP可以用作监测特定C的有效和敏感的报告基因。新形成基因的体外表达和GFP报告基因构建物可用于鉴定新基因或表征在感染过程中表达受到调控的已知基因。

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