短发夹RNA干扰c-myc重组腺病毒的构建

Construction of recombinant adenovirus mediated short hairpin RNA silence on c-myc

背景:RNA干扰已被多次证实是一种特异、高效、经济的使基因表达受抑的技术手段。目的:以短发卡RNA腺病毒表达载体为介导,构建介导shRNA-c-myc复制缺陷型重组腺病毒。设计、时间及地点:单一样本观察,于2006-09/2007-09在环境与疾病相关基因教育部重点实验室完成。材料:人骨肉瘤细胞系OS-9901由解放军第四军医大学唐都医院全军骨肿瘤研究所范清宇教授惠赠。方法:设计有短发夹RNA结构的3对单链寡核苷酸(ssoligo),经变性退火为双链寡核苷酸(dsoligo)插入穿梭质粒pENTR/U6载体,测序正确后经阳离子脂质体介导入人骨肉瘤细胞系OS-9901,通过反转录-聚合酶链反应方法筛选对c-myc沉默效果最佳穿梭质粒pENTR/U6-shRNA,然后与腺病毒骨架质粒行同源重组,筛选出正确重组子,用293A细胞包装出表达c-myc-shRNA的复制缺陷型重组腺病毒。主要观察指标:①观察细胞病变效应。②通过50%组织培养感染剂量测定病毒滴度。结果:电泳验证后的dsoligo插入穿梭质粒pENTR/U6载体,测序结果提示所构建的pENTR/U6-shRNA质粒正确。对c-myc沉默效果最佳的穿梭质粒pENTR/U6-shRNA,经PacⅠ酶切线性化后同源重组后成功构建了介导c-myc-shRNA复制缺陷型重组腺病毒。3d开始出现细胞病变效应,6d更加明显,测定证实病毒滴度为10-3.8/0.1mL。结论:成功构建介导shRNA-c-myc复制缺陷型重组腺病毒。

BACKGROUND： RNA interference has been proven to be a specific, high efficacy and economic technique to inhibit gene expression. OBJECTIVE： To design and construct the replication-incompetent recombinant adenovirus mediated shRNA （short hairpin RNA）, which was transduced into human osteosarcoma cell line （OS-9901） to silence the c-myc factor. DESIGN, TIME AND SETTING： Single-sample observation was performed at the Key Laboratory of Environment and Disease-related Gene, Ministry of Education from September 2006 to September 2007. MATERIALS： Human osteosarcoma cell line （OS-9901） was graciously provided by Professor Fan, Institute of Osteoscarcoma, Tangdu Hospital of Fourth Medical University of Chinese PLA. METHODS： Three pairs of complementary single-stranded oligonucleotides （ss oligo） containing shRNA were designed and synthesized, and then they were annealed to create a double-stranded oligonucleotide （ds oligo）. The ds oligos were cloned into pENTR/U6 vector to produce the shuttle plasmid pENTR/U6-shRNA, which was transduced into osteosarcoma cells by liposome after sequencing. The plasmid with good silence effect was selected by RT-PCR to perform the LR recombination reaction to the adenovirus backbone plasmid. The expression clone was transfected into HEK293A cell to produce replication-incompetent recombinant adenovirus mediated shRNA against c-myc. MAIN OUTCOME MEASURES： Cytopathic effect was observed and virus titer was measured by 50 percent tissue culture infective dose. RESULTS： Ds oligo validated by electrophoresis was inserted pENTR/U6-shRNA shuttle plasmid that was constructed and confirmed correct by sequencing. The optimal plasmid with good silence effect was chosen by RT-PCR from three pairs of double-stranded oligonucleotide, by Pac Ⅰ enzyme, the linearized replication-incompetent recombinant adenovirus mediated shRNA was constructed to perform the LR recombination reaction to the adenovirus backbone plasmid. The cytopathic effect of adenovirus mediated shRNA was observed since the third day and obviously on the sixth day. The titer was 10 ^-3.8/0.1 mE by 50 percent tissue culture infective dose. CONCLUSION： The recombinant adenovirus mediated shRNA against c-myc was constructed successfully.