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Optimizing the Puromycin Selection Step in our ReproRNA™-OKSGM Workflow

ReproRNA™-OKSGM is a non-integrating, self-replicating RNA-based reprogramming vector. The vector contains a puromycin-resistance cassette which allows for the selection of cells that have taken up the vector. Therefore, in the presence of puromycin, cells that do not contain the vector will die. The selection stage of the protocol starts the day after transfection and continues for 5 days in an effort to remove untransfected fibroblasts from the culture. At STEMCELL Technologies, the optimal puromycin concentration used during the selection phase was found to be 0.8 μg/mL. The aim of the puromycin selection phase of the protocol is to use enough puromycin to be cytotoxic to non-transfected fibroblasts thus reducing background, without using so much that would also be cytotoxic to transfected cells, which could result in a lower than expected reprogramming efficiency. If you observe very few cells after selection, or do not observe significant cell death by the end of the selection phase, 0.8 μg/mL of puromycin may not be the optimal concentration for your particular cell line. To determine an optimal concentration a cytotoxicity assay with puromycin should be performed.

Follow the steps below to perform a cytotoxicity assay to determine the optimal puromycin concentration to use in the ReproRNA™-OKSGM experiment.

  1. Plate fibroblasts at the same passage that will be used to perform the ReproRNA™-OKSGM experiment with at 5 x 10^4 cells/mL in Fibroblast Culture Medium (High glucose DMEM containing 10% (v/v) FBS, 1% (v/v) MEM Non-Essential Amino Acids and 2 mM L-Glutamine) onto a 96- well plate and and incubate overnight at 37°C, 5% CO2 to allow them to adhere. An example plate plan is given below, but feel free to perform as many concentration points and replicate wells as necessary.
  2. The next day, prepare sufficient aliquots of Fibroblast Culture Medium with puromycin at the various concentrations determined in Step 1. For example, prepare standards of 1.2, 1.0, 0.8, 0.6, 0.4, 0.2, 0.1 and 0 μg/mL puromycin.
  3. Examine the 96-well plate under the microscope and ensure that the fibroblasts have adhered to the surface of the plate. The cell density should be approximately 80% at this point.
  4. Aspirate the medium from all wells and replace with Fibroblast Culture Medium containing the different concentrations of puromycin. Incubate the plate overnight 37°C, at 5% CO2.
  5. Repeat steps 2 to 4 for a total of 5 days. NOTE: It is important to make Fibroblast Culture Medium containing puromycin fresh each day during this protocol. Preparing a batch for the entire duration of the cytotoxicity assay is not advised.
  6. On Day 5, determine the minimum concentration required to kill 80-100% of the cells by using a viability assay such as MTT or LDH release. Use this concentration of puromycin during the selection stage in the subsequent ReproRNA-OKSGM experiment.

Example 96-well plate plan containing triplicate assay conditions:

A\1 2 3 4 5 6 7 8 9 10 11 12
B µg/mL Puromycin
C Media
Alone
1.2 1.0 0.8 0.6 0.4 0.2 0.1 Solvent
Control
D Media
Alone
1.2 1.0 0.8 0.6 0.4 0.2 0.1 Solvent
Control
E Media
Alone
1.2 1.0 0.8 0.6 0.4 0.2 0.1 Solvent
Control
F
G
H

If you have any further questions about ReproRNA™-OKSGM, please feel free to contact Product and Scientific Support at techsupport@stemcell.com.