Mission short hairpin RNA (shRNA) lentiviral vectors expressing five ATR or p62-specific shRNAs (constructs 1 to 5) were purchased from Sigma (Sigma-Aldrich, St. Louis, MO). The vectors were packaged into capsids in 293T cells, and lentiviral particles were prepared as previously described (44 (link)). CIN612 HPV-positive cells were then transduced with these lentiviral soups overnight at 37°C. The viral soups consist of concentrated shRNA or scrambled shRNA lentiviruses and 4 μg/ml hexadimethrine bromide (Polybrene; Sigma-Aldrich, St. Louis, MO) in 5 ml E medium. The incubated cells were changed to fresh E medium the next day for an additional 48 h before further processing or analysis. Stable knockdown lines were selected by coexpressed puromycin drug resistance markers.
Mission short hairpin rna shrna lentiviral vectors
Manufactured by Merck Group
Mission short hairpin RNA (shRNA) lentiviral vectors are a laboratory tool used for gene knockdown studies. They utilize a lentiviral delivery system to introduce short hairpin RNA sequences into target cells, which can silence the expression of specific genes.
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4 protocols using mission short hairpin rna shrna lentiviral vectors
1
Generating Stable Knockdown Cell Lines
2
Lentiviral-Mediated TopBP1 Knockdown
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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.
3
Lentiviral Knockdown of TopBP1 in CIN612 Cells
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Mission short hairpin RNA (shRNA) lentiviral vectors were purchased from Sigma-Aldrich, St. Louis, MO. Lentiviral particles expressing five TopBP1-specific shRNAs (constructs 1 to 5) were prepared as previously described (22 (link)). CIN612 cells were then incubated with 5 ml fresh E medium containing concentrated TopBP1 shRNA or scrambled shRNA lentiviral soup in the presence of 4 µg/ml hexadimethrine bromide (Polybrene; Sigma-Aldrich, St. Louis, MO) overnight at 37°C. The culture medium was changed, and the transduced cells were cultured in fresh E medium for an additional 48 h before analysis. The shRNA against STAT-5 has been tested and applied in our previous work (22 (link)).
4
Lentiviral-Mediated TopBP1 Knockdown
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