Platinum e plat e retroviral packaging cell line

The amino-terminal FLAG-tagged coding sequence (CDS) of mouse Etv5 was amplified by PCR, initially cloned into T-blunt vector (SolGent), and then verified by sequencing. A confirmed Etv5 CDS fragment digested by XhoI/HpaI was sub-cloned into MigR1 retroviral vector (MigR1-ETV5-GFP). The shRNA expression vectors for knockdown of mouse Etv5 and Maf were generated using MSCV-LTRmiR30-PIG (LMP) vector (Open Biosystems) according to the manufacturer’s instruction. The target sequences were as follows. For shETV5: 5′-ACCCGAGAGACTGGAAGGCAAA-3′ and for shMAF: 5′-AAGATATAACCTGCAAGCATAT-3′.
Viruses were generated by transient co-transfection of Platinum-E (Plat-E) retroviral packaging cell line (Cell Biolabs) with the cloned retroviral vectors and pCL-Eco helper plasmid (Imgenex). Briefly, 0.5–0.8 × 10⁶ plat-E cells were plated in 6-well plates. On the next day, the cells were transfected with 1.2 μg of retroviral vector and 0.8 μg of pCL-Eco using FuGENE HD transfection reagent (E2311, Promega). Retrovirus-containing supernatants were collected 48 h later and frozen at −80 °C.

Murine Klf5 cDNA was cloned into the retroviral backbone pMX-GFP (Cell Biolabs, San Diego, CA) to produce pMX-Klf5-GFP (RV-Klf5), after which the retroviruses were packaged into the Platinum-E (PLAT-E) Retroviral Packaging Cell Line (Cell Biolabs) according to the manufacturer’s instructions. Retroviral infection was accomplished at 37°C overnight using PLAT-E supernatant supplemented with 4 µg/ml polybrene.