MISSION short hairpin RNA (shRNA) lentiviral vectors (Sigma-Aldrich, St. Louis, MO) expressing five TopBP1-specific shRNAs (constructs 1–5) were transfected into 293T cells and lentiviral particles were prepared as previously described 25 (link). CIN612 cells were then incubated with concentrated TopBP1 shRNA or scramble shRNA lentiviral soup with 4 μg/ml hexadimethrine bromide (Polybrene; Sigma-Aldrich, St. Louis, MO) in 5ml total volume E media overnight at 37°C. The cells were washed the next day, and maintained in fresh E media for an additional 48 hours before further processing or analysis. Similar processes applied to the preparation of p73 knockdown cells.
Protocol detail
Lentiviral-Mediated TopBP1 Knockdown
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Corresponding Organization : Northwestern University
Other organizations : Women's Hospital, School of Medicine, Zhejiang University, University of Chicago
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Variable analysis
independent variables
- TopBP1 shRNA (constructs 1-5)
- Scramble shRNA
- P73 knockdown
- TopBP1 expression
- Cell phenotypes or responses
- Hexadimethrine bromide (Polybrene) concentration (4 μg/ml)
- E media culture conditions
- Scramble shRNA as a negative control for TopBP1 knockdown
- No specific positive control mentioned
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