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pGB expression vectors contain the human U6 RNA polymerase III promoter, which directs constitutive, high-level expression of short RNA transcripts in many cells. Each vector also contains the neomycin/kanamycin-resistance gene to provide kanamycin resistance in bacteria and the G418 resistance in mammalian cells.
The siRNA vectors have been tested in HeLa or 293 cell lines and the knockdowns were analyzed by Western blot analysis.
The pGB siRNA vectors can be transfected into mammalian cells using Lipofectamine (Invitrogen). For transient transfection, cells can be analyzed in 24-96 hours following transfections, by Western blot analysis or other detection means. For stable transfections, cells can be selected in G418 selection medium to obtain stable cell lines with the specific gene blocked.
RNA Interference Mechanism
Once processed, the vector expressed siRNA is incorporated into a multi-protein nuclease complex known as the RNA-induced silencing complex (RISC). Homology between the sense portion of the siRNA sequence and the mRNA enables the nuclease enzyme to bind and cleave the caspase transcript into small pieces, which are degraded by the cell's machinery.
Features and Positions:
Human U6 Promoter: 1-256
Multiple cloning Site: 259-285
3' Primer: 398-426 (GAAGCATTTATCAGGGTTATTGTCTCATG)
SV40 Promoter: 470-808
Neomycin / Kanamycin Resistance ORF: 843-1634
5' Primer: 2789-2813 (CGTCGATTTTTGTGATGCTCGTCAG)
pUC Origin of Replication: 2222-3003
Lane 1: Control siRNA
Lane 2: Caspase 3 siRNA
Caspase 3 siRNA vector ab12490 blocks caspase expression in HeLa cells. The caspase 3 siRNA Vector (ab12490) was transfected into HeLa cells using Lipofectmine (Invitrogen). The control vector (ab12505) with a siRNA sequence that has no homology to mammalian gene was also transfected as a negative control. The western blot was probed with a caspase-3 polyclonal antibody.
ab12490 has not yet been referenced specifically in any publications.