Cloning, mutagenesis, and deletion of DISC1 and Kal-7 were conducted as described in the prior publication25 (link). Wild-type, constitutively-active (Rac1-CA, G12V), and dominant-negative form of Rac1 (Rac1-DN, T17N) were from Missouri University of Science and Technology (Rolla, MO). H1-RNA polymerase III promoter-driven shRNA against DISC1 (#1 with strong effect and #2 with weaker effect), which also carry EGFP under the downstream of CMV promoter, were previously described25 (link). Cultured cells were transfected with plasmids by the use of LipofectAMINE2000 (Invitrogen). In primary neuron culture, 2μg of pSuper-Venus RNAi were transfected into 1.5 × 105 cells. For 293 cells, 2 μg of pRK/DISC1-HA expression construct and 2 μg of pSuper-Venus RNAi construct were transfected (at 90 % confluence in 18 mm dish). In standard analyses, primary neurons were transfected after 23 DIV and maintained for 1–6 days after transfection.
Protocol detail
Genetic Manipulation of Neural Signaling Pathways
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Cells
Cultured cells
Deletion
Dish
Mutagenesis
Neurons
Plasmids
Rna polymerase iii
Rnai
Shrna
Transfection
Corresponding Organization :
Other organizations : Johns Hopkins Medicine, Johns Hopkins University, University at Buffalo, State University of New York, University of Glasgow, Northwestern University, City of Hope, Pfizer (United States)
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Variable analysis
independent variables
- Wild-type Rac1
- Constitutively-active Rac1 (Rac1-CA, G12V)
- Dominant-negative Rac1 (Rac1-DN, T17N)
- H1-RNA polymerase III promoter-driven shRNA against DISC1 (#1 with strong effect and #2 with weaker effect)
- Not explicitly mentioned
- Transfection of plasmids using LipofectAMINE2000
- Transfection of 2μg of pSuper-Venus RNAi into 1.5 × 10^5 primary neuron cells
- Transfection of 2 μg of pRK/DISC1-HA expression construct and 2 μg of pSuper-Venus RNAi construct into 293 cells at 90% confluence in 18 mm dish
- Timing of primary neuron transfection (after 23 DIV) and maintenance period (1–6 days after transfection)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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