Protocol for Making Retroviral Reprogramming Factors

This protocol was developed as an efficient and consistent method to produce functional retroviral reprogramming factors. It avoids the need for concentrating virus via ultracentrifugation.

• Grow 293T cells in a T175 flask. Cells should be 85% confluent.
• Aspirate 293T media from each 293T flask. Feed each flask with 16 ml of fresh 293T media.
• Make 2 sets of 4×50 ml conical tubes.
• For the first set of 4×50 ml conical tubes. Give each tube 8 ml of OPTI-MEM media and 127.3 ul of Lipofectamine 2000. Flick each 50 ml conical tube and let sit for 5 minutes at room temperature.
• For the second set of 4×50 ml conical tubes. Label each tube with a different viral factor (Oct4, Sox2, klf4, or c-myc). Each tube gets 8 ml of OPTI-MEM, 32ug of gag-pol, 12.7 μg of vsvg, and 47.7ug of the appropriate viral plasmid.
• Combine one tube of the first set (OPTI-MEM + Lipofectamine 2000) with 1 tube of the second set (OPTI-MEM + gag-pol + vsvg + viral plasmid) for each factor. Each tube will now have 16 ml total in it.
• Mix each tube and let sit for 20 minutes at room temperature.
• Add the appropriate tube of 16 ml mixture to its correspondingly labeled T175 flask. Place in a 37°C incubator.
• After 16 hours, change media and replace with 32 ml fresh 293T media.
• 24 hours later, collect the 32 ml of 293T media from each T175 flask and filter. This media is now virus-rich.
• Aliquot virus and freeze at -80°C freezer.

• A least 1 T175 flask per factor will be needed, so you must have at least 4 flasks. Each T175 should be fed with 32 ml of 293T media.
• In general, we like to combine the four factors and aliquots into 4 ml. Each aliquot is then ready to reprogram one well of a 6-well plate when using our infection protocol. The virus can be stored at -80°C for at least 6 months. Avoid repeated freeze-thaw cycles with the virus.

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