All lentiviruses were packaged in HEK293T/17 cells according to published protocols (54). Briefly, 293T cells were transiently transfected with psPAX2, MD2.G, and pDEST673 vector, pDEST673/WT ERα, or pDEST673/Q375H mutant carrying the neomycin resistance gene using Lipofectamine 2000. Supernatant was collected 48 hours after transfection and concentrated by centrifugation at 50,000g for 2 hours over a 20% sucrose cushion. Pellets were resuspended in PBS and used for infection. Titers were determined using qPCR to measure the number of lentiviral particles integrated into the transduced HEK293T genome. Multiplicity of infection (MOI) ranging from 25 to 180 was used for infection of HepG2 cells. After 3 days of infection, cells were selected with geneticin (1.2 mg/ml, Invitrogen, #11811-031) and a stably pooled population of cells was obtained after 2 weeks. Stable integration of ERα protein was detected by Western blot (fig. S2A). Despite our best efforts, we were only able to isolate HepG2/Q375H tranfectants that expressed higher levels of mutant ERα than WT ERα.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.