Research ArticleResearch Article

Construction and Identification of a Vector Expressing RNA Interference Aimed at the Human CyclinD1 Gene and its Expression in Vitro

Dongmei Yu, Lijun Hao, Ying Li, Lihong Ren and Ying Liu
Chinese Journal of Clinical Oncology October 2007, 4 (5) 338-342; DOI: https://doi.org/10.1007/s11805-007-0338-7

Article Figures & Data

Figures

  • Identification of recombinant plasmids by restriction endonuclease digestion. M: DL2000 marker; 1~4:pSUPER-C1~C4; 5: pSUPER cut with EcoR I/Hind III, respectively.
    Fig.1.

    Identification of recombinant plasmids by restriction endonuclease digestion. M: DL2000 marker; 1~4:pSUPER-C1~C4; 5: pSUPER cut with EcoR I/Hind III, respectively.

  • Morphology and expression of EGFP in HO-8910 cells after transfection (200×).A: control cells; B: Trans-fected with pSUPER-C2; C: Transfected with pSUPER-C2 (by fluorescence microscope).
    Fig.2.

    Morphology and expression of EGFP in HO-8910 cells after transfection (200×).A: control cells; B: Trans-fected with pSUPER-C2; C: Transfected with pSUPER-C2 (by fluorescence microscope).

  • Expression of cyclinD1 mRNA in HO-8910 cells transfected with pSUPER-C detected by RT-PCR(β-actin 583 bp product was used as a control). M:DL2000 marker; 1:Control cells;2: Transfected with pSUPER; 3~4: Transfected with pSUPER-C1; 5~6: Transfected with pSUPER-C2; 7~8: Transfected with pSUPER-C3; 9~10: Transfected with pSUPER-C4 HO-8910 cells.
    Fig.3.

    Expression of cyclinD1 mRNA in HO-8910 cells transfected with pSUPER-C detected by RT-PCR(β-actin 583 bp product was used as a control). M:DL2000 marker; 1:Control cells;2: Transfected with pSUPER; 3~4: Transfected with pSUPER-C1; 5~6: Transfected with pSUPER-C2; 7~8: Transfected with pSUPER-C3; 9~10: Transfected with pSUPER-C4 HO-8910 cells.

  • Quantitation of the inhibitory percentage of cyclinD1 mRNA in transfected HO-8910 cells. Each level of PCR product of cyclinD1 gene was quantified and normalized to the level of β-actin. The inhibitory rate was calculated by comparing to the control cells. The results were expressed as mean±SD from independent experiments.
    Fig.4.

    Quantitation of the inhibitory percentage of cyclinD1 mRNA in transfected HO-8910 cells. Each level of PCR product of cyclinD1 gene was quantified and normalized to the level of β-actin. The inhibitory rate was calculated by comparing to the control cells. The results were expressed as mean±SD from independent experiments.

Tables

  • Table 1.

    Oligonucleotides corresponding to the target sequence and the control sequence

    SiRNASequenceLocation
    C15’-GATCCCCAAACAGATCATCCGCAAATTCAAGAGATTTGCGGATGATCTGTTTGTTTTTTGGAAA-3’
    5’-AGCTTTTCCAAAAAACAA ACAGATCATCCGCAAATCTCTTGAATTTGCGGATGATCTGTTTGGGG-3’    		496-514
    C25’-GATCCCACACGCGCAGACCTTCGAGTTCAAGAGATAACGAAGGTCTGCGCGTGTTTTTTGGAAA-3’
    5’-AGCTTTTCCAAAAAAACACGCGCAGACCTTCGTTTCTCTTGAATCCGAAGGTCTGCGCGTGTGGG-3’    		576-594
    C35’-GATCCCGCATGTTCGTGGCCTCTAATTCAAGAGATTAGAGGCCACGAACATGCTTTTTTGGAAA-3’
    5’-AGCTTTTCCAAAAAAGCATGTTCGTGGCCTCTAATCTCTTGAAGTAGAGGCCACGAACATGCGGG-3’    		729-747
    C45’-GATCCCGCATGGTGATAGGAAACAGTTCAAGAGATTAGAGGCCACGAACATGCTTTTTTGGAAA-3’
    5’-AGCTTTTCCAAAAAAGCATGTTCGTGGCCTCTACTCTCTTGAATAGAGGCCACGAACATGACGGG-3’    		934-952
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