Smulian A George, Gibbons Reta S, Demland Jeffery A, Spaulding Deborah T, Deepe George S
Infectious Disease Division, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA.
Eukaryot Cell. 2007 Nov;6(11):2066-71. doi: 10.1128/EC.00139-07. Epub 2007 Sep 14.
The Escherichia coli hygromycin phosphotransferase (hph) gene, which confers hygromycin resistance, is commonly used as a dominant selectable marker in genetically modified bacteria, fungi, plants, insects, and mammalian cells. Expression of the hph gene has rarely been reported to induce effects other than those expected. Hygromycin B is the most common dominant selectable marker used in the molecular manipulation of Histoplasma capsulatum in the generation of knockout strains of H. capsulatum or as a marker in mutant strains. hph-expressing organisms appear to have no defect in long-term in vitro growth and survival and have been successfully used to exploit host-parasite interaction in short-term cell culture systems and animal experiments. We introduced the hph gene as a selectable marker together with the gene encoding green fluorescent protein into wild-type strains of H. capsulatum. Infection of mice with hph-expressing H. capsulatum yeast cells at sublethal doses resulted in lethality. The lethality was not attributable to the site of integration of the hph construct into the genomes or to the method of integration and was not H. capsulatum strain related. Death of mice was not caused by altered cytokine profiles or an overwhelming fungal burden. The lethality was dependent on the kinase activity of hygromycin phosphotransferase. These results should raise awareness of the potential detrimental effects of the hph gene.
赋予潮霉素抗性的大肠杆菌潮霉素磷酸转移酶（hph）基因，在转基因细菌、真菌、植物、昆虫和哺乳动物细胞中常用作显性选择标记。很少有报道称hph基因的表达会产生预期之外的影响。潮霉素B是在荚膜组织胞浆菌基因敲除菌株的产生或作为突变菌株的标记物的分子操作中最常用的显性选择标记。表达hph的生物体在长期体外生长和存活方面似乎没有缺陷，并已成功用于短期细胞培养系统和动物实验中研究宿主-寄生虫相互作用。我们将hph基因作为选择标记与编码绿色荧光蛋白的基因一起导入荚膜组织胞浆菌的野生型菌株中。用亚致死剂量的表达hph的荚膜组织胞浆菌酵母细胞感染小鼠会导致死亡。这种致死性并非归因于hph构建体整合到基因组中的位点或整合方法，也与荚膜组织胞浆菌菌株无关。小鼠死亡不是由细胞因子谱改变或真菌负荷过重引起的。致死性取决于潮霉素磷酸转移酶的激酶活性。这些结果应提高人们对hph基因潜在有害影响的认识。
