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  • Title: [Construction of recombinant adenovirus vector by c-myc silencing].
    Author: Wang D, Liu M, Wang M, Zhang Y.
    Journal: Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi; 2008 Aug; 22(8):969-73. PubMed ID: 18773816.
    Abstract:
    OBJECTIVE: 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. METHODS: 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). RESULTS: 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. CONCLUSION: The recombinant adenovirus mediated shRNA c-myc is constructed in vitro through RNA interference technology.
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