The retrovirus was generated by transfecting WSX1-IRES-GFP, WSX1-MUT-DSRed-IRES-GFP, or control GFP and DsRed constructs into Phoenix Eco packaging cells.
The Phoenix Eco packaging cells were transfected with pLNCX2-EGFP using calcium phosphate-based transfection.
High-titer, replication-in-competent retroviral particles were produced in Phoenix Eco packaging cell line (Orbigen, San Diego, CA).
High-titre, replication-incompetent retroviral particles encoding the RNA of interest were produced in the Phoenix Eco packaging line (Orbigen, San Diego, CA, USA) for murine melanocytes.
For stable transduction, retroviruses were produced in Phoenix Eco packaging cells, following the Nolan Lab protocol (http://www.stanford.edu/group/nolan/protocols/pro_helper_dep.html).edu/group/nolan/protocols/pro_helper_dep.html
Virus-producing RetroPack PT67 cells were established through a ping-pong method, which uses the virus from the Eco packaging cell line to infect RetroPack PT67 cells.
All cell lines, the mouse fibroblast cell line (NIH/3T3), 293T human renal epithelial cell, RetroPackPT67 packaging cells and Phoenix Eco packaging cells were purchased from ATCC (Rockville, MD, USA) and grown in Dulbecco's modified eagle medium (DMEM; GIBCO, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA), 100 IU/ml penicillin and 100 μg/ml streptomycin.
I do like their eco-packaging, though.
Retroviral supernatants from of Phoenix-Eco packaging cells cultured at 37°C were collected after 48 h.
The pFB-hDicer construct was introduced into 293T cells together with the pCL-Eco packaging plasmid by calcium phosphate transfection.
High-titre viruses were produced by transient transfection of retroviral constructs into the Phoenix-Eco packaging cell line using FuGENE HD transfection reagent (Roche) according to standard procedures.
