To generate the lentiviruses, envelope plasmid, packaging plasmid (Open Biosystems Inc.) and shRNA expressing plasmid were co-transfected into HEK293FT cells (Invitrogen). Lentiviral short hairpin RNA (shRNA) expression vector for mTOR and the control pLKO.1 plasmid were purchased from Addgene. Lentiviral tet-on-driven short hairpin RNA (shRNA) expression vector for HSF1 and the control tripZ plasmid were purchased from Open Biosystems Inc. Lentiviral shRNA expression and control vectors for HuR and the HuR overexpression plasmid 1A3-HuR were kind gifts from Dr. Michael Sherman (Boston University) 13. Virus-containing medium was collected 48 and 72 hr after transfection. MDA-MB-231 cells were infected by incubation with the lentivirus-containing medium and thereafter the cells were treated with puromycin for selection of knockdown cells. Cells overexpressing HuR were positively selected with G418 (Neomycin, 500ug/ml). For induction of shRNA targeting HSF1, MDA-MB-231 cells were treated with 2μg/ml doxycycline. The HSF1-GFP fusion expression plasmid construct used in the study was prepared in pEGFP-N3 vector.
Packaging plasmid
Manufactured by Thermo Fisher Scientific
Packaging plasmid is a circular DNA molecule used to package recombinant DNA into viral particles for gene delivery applications. It contains the necessary genetic elements for packaging the DNA cargo into a viral capsid, enabling efficient transduction of target cells.
Automatically generated - may contain errors
2 protocols using packaging plasmid
1
Lentiviral Knockdown and Overexpression
2
Lentiviral Knockdown and Overexpression
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To generate the lentiviruses, envelope plasmid, packaging plasmid (Open Biosystems Inc.) and shRNA expressing plasmid were co-transfected into HEK293FT cells (Invitrogen). Lentiviral short hairpin RNA (shRNA) expression vector for mTOR and the control pLKO.1 plasmid were purchased from Addgene. Lentiviral tet-on-driven short hairpin RNA (shRNA) expression vector for HSF1 and the control tripZ plasmid were purchased from Open Biosystems Inc. Lentiviral shRNA expression and control vectors for HuR and the HuR overexpression plasmid 1A3-HuR were kind gifts from Dr. Michael Sherman (Boston University) 13. Virus-containing medium was collected 48 and 72 hr after transfection. MDA-MB-231 cells were infected by incubation with the lentivirus-containing medium and thereafter the cells were treated with puromycin for selection of knockdown cells. Cells overexpressing HuR were positively selected with G418 (Neomycin, 500ug/ml). For induction of shRNA targeting HSF1, MDA-MB-231 cells were treated with 2μg/ml doxycycline. The HSF1-GFP fusion expression plasmid construct used in the study was prepared in pEGFP-N3 vector.
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