Macrophage lines expressing shAkt or controls were generated by transduction of SMARTvector Inducible Lentiviral shRNA system (Dharmacon, Lafayette CO). Custom made lentiviruses that included one of three sequences: shAkt1: ATCGGAAGTCCATCGTCTC, shAkt2: GGGACTCTCGCTGATCCAC shAkt3: CGTTTGTGCAGCCAGCCCT were developed. A control Lentiviral recombinant containing the sequence TGGTTTACATGTTGTGTGA was also used to generate a control cell line. Lentiviral particles prepared by Dharmacon were used to transduce RAW264.7 macrophages. After 24 hr, transduced macrophages were selected by growth in 6ug/ml puromycin in complete DMEM at 37° C under 5% CO2 following protocols from Dharmacon. After at least 2 passages under antibiotic selection, each cell line was then assessed for Akt shRNA expression. The expression of Akt shRNA was induced by growth in 1uM doxycycline.
Smartvector inducible lentiviral shrna system
Manufactured by Horizon Discovery
The SMARTvector Inducible Lentiviral shRNA system is a laboratory tool designed for the controlled expression of short hairpin RNA (shRNA) in target cells. It utilizes a lentiviral vector system to enable stable integration and inducible regulation of shRNA expression.
Automatically generated - may contain errors
2 protocols using smartvector inducible lentiviral shrna system
1
Inducible Akt Knockdown in Macrophages
2
Generating Inducible Cell Lines Expressing Proteins
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Inducible Flp-In T-REx 293 (ThermoFisher) stable cell lines expressing Strep-tag (ST)/HA affinity tagged GRSF1 and DHX36 were generated by transfection of pcDNA5/FRT/TO vector as per manufacturers protocol (ThermoFisher). Cells were selected with Blasticidin (Gibco) and Hygromycin D (Sigma). For shRNA knockdowns, SMARTvector inducible lentiviral shRNA system (Dharmacon) was used to generate viral particles. Transduction efficiency was optimised to 30% to ensure single incorporation events and hairpin expressing cells selected as per manufacturers’ instructions. Protein knockdowns were achieved by treating cells with 1 μg/ml Doxycycline for 72 h.
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