Retrovirus was packaged in Phoenix cells as previously described 17 (link). Briefly, Phoenix packaging cells (ATCC CRL-3213) were co-transfected with 10 μg of the pMSCV overexpression plasmids (pMSCV-Zfp423-WT, pMSCV-Zfp423-ΔNID or empty vector) and 5 μg gag-pol and 5 μg VSV-g plasmids using Lipofectamine LTX (ThermoFisher Scientific, #15338100). Subsequently, FIPs were transduced with diluted virus (1:10 ratio) in ITS media containing 8 μg/ml polybrene (Sigma, #TR-1003) for 24 hours. Following transduction, cells were maintained in ITS media for ≥ 48 hours before beginning treatments.
Protocol detail
Retroviral Transduction of FIPs
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Other organizations : The University of Texas Southwestern Medical Center
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Variable analysis
independent variables
- Zfp423-WT overexpression
- Zfp423-ΔNID overexpression
- Empty vector
- FIPs transduced with diluted virus
- Phoenix packaging cells (ATCC CRL-3213)
- ITS media
- Polybrene (8 μg/ml)
- Positive control: Phoenix packaging cells co-transfected with gag-pol and VSV-g plasmids
- Negative control: Empty vector
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