What is lentiviral shRNA?
Lentiviral vectors provide a means to express short hairpin RNA (shRNA) to induce stable and long-term gene silencing in both dividing and non-dividing cells and thus, are being intensively investigated for this purpose.
How do you transduce lentivirus?
Lentiviruses (a genus of retrovirus) express reverse transcriptase, which converts the viral RNA to double stranded DNA, and integrase, which inserts this viral DNA into the host DNA. Once the viral DNA is integrated into the host DNA, it divides along with host cell and none are the wiser.
How do you transfect shRNA?
For each transfection, add 0.8 ml shRNA Plasmid Transfection Medium to well. Add the 200 µl shRNA Plasmid DNA/shRNA Plasmid Transfection Reagent Complex (Solution A + Solution B) dropwise to well, covering the entire layer. Gently mix by swirling the plate to ensure that the entire cell layer is immersed in solution.
What is inducible shRNA?
TRIPZ Inducible Lentiviral shRNA combines the advantages of microRNA-adapted lentiviral vector design with Tet-inducibility to generate an RNAi trigger that permits reversible, controlled gene silencing. The vector is engineered to be Tet-On® such that shRNA expression is induced in the presence of doxycycline.
What are lentiviral particles?
Lentiviral particles are generated by co-transfection of the lentiviral expression vector with lentiviral packaging plasmids into e.g. HEK293 cells and harvesting of the virus-containing supernatant afterwards. Pseudoviral particles can be concentrated, frozen, and used in later experiments.
How long does it take shRNA to work?
Duration of siRNA silencing. In general, the earliest time after which the silencing effect can be observed is 24 hours. It retains cell type dependent for 4–7 days.
How is shRNA processed?
If the stem of an shRNA is longer than 20 bp, the shRNA is processed by Dicer. If the stem of an shRNA is 19−20 bp, Dicer and Ago2 compete to process the shRNA. For an shRNA containing a stem between 16 and 18 bp, it is only processed by Ago2.
How do you use shRNA lentiviral particles to infect cells?
Infect cells by adding the shRNA Lentiviral Particles to the culture. Swirl the plate gently to mix and incubate overnight. The amount of viral particles to use varies greatly depending on the characteristics of the cell line used. NOTE: Keep thawed shRNA Lentiviral Particles on ice.
How do I produce viral stocks from Mir RNAi or shRNA?
Once you have your miR RNAi or shRNA sequence in a lentiviral or adenoviral backbone, producing the viral stocks follows the same protocols for both vector systems. Table 2. Choose a lentiviral or adenoviral RNAi system. For Research Use Only.
How do I express the shRNA of interest in lenti4/block-it™-DEST?
To express the shRNA of interest in a tetracycline-regulated manner, use the TetR-expressing cell line as the host for your Lenti4/BLOCK-iT™-DEST lentiviral construct. After transduction, you have two options to express the shRNA of interest:
What are the lentiviral and packaging vectors used in block-it™ inducible H1 lentiviral RNAi?
The lentiviral and packaging vectors supplied in the BLOCK-iT™ Inducible H1 Lentiviral RNAi System are third-generation vectors based on lentiviral vectors developed by Dull et al., 1998.