To overexpress HIF‐1α, cells were transfected with pEF‐BOS‐human HIF‐1α or pEF‐BOS (control) using Lipofectamine LTX (Invitrogen). To suppress HIF‐1α expression, we used the MISSION shRNA lentiviral transduction system (TRCN0000003810; Sigma‐Aldrich).20 GFP‐expressing Aldefluorpos cells were established using MISSION TurboGFP Control Particle (SHC003V; Sigma‐Aldrich) as control cells. And, to suppress HIF‐2α expression, cells were transfected with either HIF‐2α siRNA or negative control siRNA (Qiagen, Basel, Switzerland), using Lipofectamine LTX (Invitrogen).21 All kits were used according to the manufacturer's instructions.
Mission shrna lentiviral transduction system
Manufactured by Merck Group
Sourced in Sao Tome and Principe
The MISSION shRNA lentiviral transduction system is a laboratory tool designed for the delivery and expression of short hairpin RNA (shRNA) in target cells. It utilizes lentiviral vectors to efficiently transduce a wide range of cell types, enabling the study of gene function through targeted knockdown.
Automatically generated - may contain errors
2 protocols using mission shrna lentiviral transduction system
1
Overexpression and Suppression of HIF-1α and HIF-2α
2
ACTN4 Knockdown Assay Using shRNA and siRNA
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Silencer negative control RNA and siRNA targeting ACTN4 (Hs_ACTN_5 and Hs_ACTN4_10) (Qiagen) were used at a final concentration of 50 nM. Transient transfection was performed using Lipofectamine 2000 (Life Technologies) in accordance with the manufacturer's protocol.
Stable knockdown was performed using the Mission shRNA lentiviral transduction system (Sigma-Aldrich, St. Louise, MO). Two shRNA constructs targeting ACTN4 (TRCN0000055784 and TRCN0000055785) and two control constructs (empty vector control SHC001V) were selected from the RNAi Consortium shRNA library. A549-luc-C8 cells were plated in 100 μL RPMI 1640 medium per well on 96-well plates and incubated overnight. The medium was then changed to growth medium containing hexadimethrine bromide (Sigma) at a concentration of 8 μg/mL to enhance transduction efficiency. After 16 h incubation with lentiviral particles, cells were selected with puromycin (Sigma) at a concentration of 10 μg/mL.
Stable knockdown was performed using the Mission shRNA lentiviral transduction system (Sigma-Aldrich, St. Louise, MO). Two shRNA constructs targeting ACTN4 (TRCN0000055784 and TRCN0000055785) and two control constructs (empty vector control SHC001V) were selected from the RNAi Consortium shRNA library. A549-luc-C8 cells were plated in 100 μL RPMI 1640 medium per well on 96-well plates and incubated overnight. The medium was then changed to growth medium containing hexadimethrine bromide (Sigma) at a concentration of 8 μg/mL to enhance transduction efficiency. After 16 h incubation with lentiviral particles, cells were selected with puromycin (Sigma) at a concentration of 10 μg/mL.
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