Wang Dongqi, Liu Miao, Wang Min, Zhang Yingang
Department of Orthopaedics, the First Affiliated Hospital, Medical College of Xi'an Jiaotong University, Xi'an Shaanxi, 710061, PR China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2008 Aug;22(8):969-73.
To design, construct and select the optimal replication-defective recombinant adenovirus mediated short hairpin RNA (shRNA) which is transduced into human osteosarcoma cells to silence c-myc gene expression, and to construct the recombinant adenovirus vector expressing c-myc-shRNA and determine its viral titer.
Three pairs of complementary single-stranded oligonucleotides (ss oligos) were designed and synthesized, and then they were annealed to create a double-stranded oligonucleotide (ds oligos).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 chosen by RT-PCR to perform the LR recombination reaction to the adenovirus backbone plasmid. The expression clone was transfected into HEK293A cells to produce replication-incompetent recombinant adenovirus mediated shRNA against c-myc whose cytopathic effect was observed and viral titer was determined by the viral particle (VP) method and 50% tissue culture infective dose (TCID50).
Ds oligos, which was verified by electrophoresis, was cloned into pENTR/U6 vector to produce pENTR/U6-shRNA shuttle plasmid, which was confirmed to be corrected by sequencing. The optimal plasmid with good silence effect was chosen by RT-PCR from the three pairs of double-stranded oligonucleotide. By Pac I enzyme, the linearization replication-defective recombinant adenovirus mediated shRNA was constructed to perform the LR recombination reaction to the adenovirus backbone plasmid. The cytopathic effect and vacuole phenomenon of adenovirus mediated shRNA appeared at 3 days and became obvious at 6 days. The adenovirus virus titer in the first generation was 5.23 x 10(9) VP/mL, and reached 2.26 x 10(12) VP/mL via 3-4 generations' amplification. The viral titer was 10(-3.8)/0.1 mL determined by VP method and TCID50.
The recombinant adenovirus mediated shRNA c-myc is constructed in vitro through RNA interference technology.
设计、构建并筛选出最佳的复制缺陷型重组腺病毒介导的短发夹RNA（shRNA），将其转导入人骨肉瘤细胞以沉默c-myc基因表达，并构建表达c-myc-shRNA的重组腺病毒载体并测定其病毒滴度。
设计并合成三对互补单链寡核苷酸（ss oligos），然后将它们退火形成双链寡核苷酸（ds oligos）。将ds oligos克隆到pENTR/U6载体中以产生穿梭质粒pENTR/U6-shRNA，测序后通过脂质体转导入骨肉瘤细胞。通过RT-PCR从具有良好沉默效果的质粒中选择进行与腺病毒骨架质粒的LR重组反应。将表达克隆转染入HEK293A细胞以产生针对c-myc的无复制能力的重组腺病毒介导的shRNA，观察其细胞病变效应并通过病毒粒子（VP）法和50%组织培养感染剂量（TCID50）测定病毒滴度。
经电泳验证的ds oligos被克隆到pENTR/U6载体中产生pENTR/U6-shRNA穿梭质粒，测序确认其正确。通过RT-PCR从三对双链寡核苷酸中选择出具有良好沉默效果的最佳质粒。通过Pac I酶构建线性化的复制缺陷型重组腺病毒介导的shRNA以进行与腺病毒骨架质粒的LR重组反应。腺病毒介导的shRNA的细胞病变效应和空泡现象在3天时出现并在6天时变得明显。第一代腺病毒病毒滴度为5.23×10⁹ VP/mL，经3至4代扩增后达到2.26×10¹² VP/mL。通过VP法和TCID50测定病毒滴度为10⁻³.⁸/0.1 mL。
通过RNA干扰技术在体外构建了重组腺病毒介导的shRNA c-myc。
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