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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
ab12504 has not yet been referenced specifically in any publications.