Millipore STEMCCA Cre-Excisable Constitutive Polycistronic (OKSM) Lentivirus Reprogramming Kit
A slight modification of the manufacturer's instructions is used. Please refer to the user manual—a brief description of the protocol is provided below with the modifications described.
- Prior to starting, determine the seeding density of your target cells. The optimal plating density is determined as the number of cells that should be plated at Day 0 in order to have the cells reach 90–95% confluency by Day 6.
- Day 0—Plate 2 wells at this density in normal growth medium (for example DMEM containing 10% FBS) and incubate overnight.
- Day 1—Trypsinize or Accutase treat one well and count. This should be used to determine the amount of virus to use on the target well
- Replace the medium on the target well with 1 mL fresh culture medium.
- Add Polybrene transfection reagent to a final concentration of 5 μg/mL and the required volume of virus. Incubate overnight.
- Day 2—Wash the cells with PBS and repeat transfection. Incubate overnight.
- Day 3—Wash cells with PBS and replace with fresh culture medium containing reprogramming chemicals (5uM PS48, Reagents Direct; 0.5uM A-83-01, StemGent; 0.25mM Sodium Butyrate, StemGent).
- Day 4–5—Replace medium daily with fresh culture medium containing reprogramming chemicals.
- Day 5—Prepare inactivated MEF feeders in 6-well plates as for normal pluripotent stem cell culture—1.5x105/well on gelatin.
- Day 6—Trypsinize or Accutase treat the transfected well and count the cell number. Plate 1.5x104 cells per well in hESC culture medium supplemented with 10ng/ml bFGF and the reprogramming chemicals. You may also add 10uM Rock inhibitor during plating (Y27632, Calbiochem).
- Day 7—Do not change the medium.
- Day 8–12—Change the medium daily with fresh hESC containing 10ng/ml bFGF and the reprogramming chemicals. NOTE: Stop adding the reprogramming chemicals when colonies start to appear.
- Day 13—Add 1.5x105 inactivated MEFs to each well of the 6-well plate containing virus-infected cells. Add fresh inactivated MEFs every 7th day to replenish older MEFs during the reprogramming timecourse.
- Continue feeding until colonies are robust. The first colonies to appear may not be fully reprogrammed and should be monitored for a further 5–6 days before picking.
- We generally pick after Day 21. Colonies are picked off under a dissecting microscope under sterile conditions and transferred to a 24-well plate prepared with MEF feeders or Matrigel in the appropriate medium—1 colony per well. If the colonies are large triturate slightly against the corner of the well.
- Do not feed the day after picking but feed every day thereafter with regular hESC medium containing 4ng/ml bFGF. Pass as appropriate.