Transcriptional activities of two newly identified Haemaphysalis longicornis tick‐derived promoter regions in the Ixodes scapularis tick cell line (ISE6)

Ticks are obligate haematophagous ectoparasites considered to be second to mosquitoes as vectors of human diseases and the most important vector for animals. Despite efforts to control tick infestations, they remain a serious health problem. Gene manipulation has been established in mosquitoes and led to the control of mosquito populations and of mosquito‐borne pathogens. Therefore, gene manipulation could be useful for controlling ticks and tick‐borne pathogens. To investigate effective gene expression vectors for ticks, the promoter activities of commercial plasmids were evaluated in a tick cell line (ISE6). Dual luciferase assays revealed that pmirGLO, the human phosphoglycerate kinase promoter contained plasmid vector, showed the highest activity in ISE6 cells amongst the tested plasmids. Moreover, we identified the promoter regions of the Haemaphysalis longicornis actin (HlAct) and the intracellular ferritin (HlFer1) genes. To construct a more effective expression vector for ticks, these promoter regions were inserted into pmirGLO (pmirGLO‐HlAct pro and pmirGLO‐HlFer1 pro). The pmirGLO‐HlAct pro vector showed significantly higher promoter activity than pmirGLO, whereas the pmirGLO‐HlFer1 pro vector demonstrated significantly lower promoter activity than pmirGLO in ISE6 cells. The HlAct promoter region may have high promoter activity in ISE6 cells. The results of the present study provide useful information for the development of a genetic modification system in ticks.     

Honey bee promoter sequences for targeted gene expression

The honey bee, Apis mellifera, displays a rich behavioural repertoire, social organization and caste differentiation, and has an interesting mode of sex determination, but we still know little about its underlying genetic programs. We lack stable transgenic tools in honey bees that would allow genetic control of gene activity in stable transgenic lines. As an initial step towards a transgenic method, we identified promoter sequences in the honey bee that can drive constitutive, tissue‐specific and cold shock‐induced gene expression. We identified the promoter sequences of Am‐actin5c, elp2l, Am‐hsp83 and Am‐hsp70 and showed that, except for the elp2l sequence, the identified sequences were able to drive reporter gene expression in Sf21 cells. We further demonstrated through electroporation experiments that the putative neuron‐specific elp2l promoter sequence can direct gene expression in the honey bee brain. The identification of these promoter sequences is an important initial step in studying the function of genes with transgenic experiments in the honey bee, an organism with a rich set of interesting phenotypes.     

转录因子E2F1调控KIR3DL1基因表达的分子机制

目的 研究转录因子E2F1对KIR3DL1基因启动子转录活性的影响,明确其调控KIR3DL1基因表达的分子机制.方法 采用PCR方法 从K562细胞基因组DNA中扩增突变型KIR3DL1基因启动子序列,将产物连接到pGL3-Basic荧光素酶载体,构建报告重组子;采用染色质免疫共沉淀方法 检测E2F1与KIR3DL1启动子在细胞内的结合;采用阳离子脂质体SuperFect包裹突变型和野生型KIR3DL1启动子-荧光素酶报告重组子,然后转染K562细胞,染色质免疫共沉淀方法 检测E2F1与重组子在细胞内的结合,双荧光素酶检测试剂盒测定荧光素酶活性;将E2F1真核表达载体与野生型KIR3DL1启动子-荧光素酶报告重组子共转染K562细胞,双荧光素酶检测试剂盒测定荧光素酶活性.结果 成功构建突变型KIR3DL1启动子-荧光素酶报告重组子,测序证实,在K562细胞KIR3DL1启动子区1个潜在的转录因子E2F1结合位点有1个自然发生的点突变(TTTGGCGC→TTCGGCGC);E2F1完全不能与K562细胞中突变型KIR3DL1启动子结合,但能与NK-92MI细胞中没有突变的野生型KIR3DL1启动子结合;突变型KIR3DL1启动子.荧光素酶报告重组子保留了部分与E2F1的结合能力,但其相对荧光素酶活性较野生型降低了50%;共转染E2F1真核表达载体使野生型KIR3DL1启动子-荧光素酶报告重组子的相对荧光素酶活性增加为对照组的2倍以上.结论 转录因子E2F1参与调控KIR3DL1基因转录激活,E2F1结合位点上CpG二核苷酸的数量和甲基化模式可能影响转录因子E2F1与靶序列的结合.