Editing His-Tat-NLS-Cre-Purification (section)

=== <br/>Cre Recombinase Transduction into Undifferentiated hES Cells ===

Culture hES cells in KO/SR medium on tissue culture plates coated with mitotically inactivated mouse embryonic fibroblasts (MEFs). Cells are passaged by manual scraping or collagenase IV treatment.

Note: Cre protein transduction into hES cells is most efficient if the colonies are very small, ideally clumps of few cells. Therefore, the hES cell colonies are dissociated using accutase II (PAA Laboratories) prior transduction. Accutase II has the advantage that it selectively detaches the hES cells from feeder cells if incubation does not exceed 30 min. The single-cell suspension is plated on MEFs again and allowed to adhere for 24 h. Within this time, very small hES cell colonies are formed, which have the optimal size for protein transduction.

Transduction efficiency is optimal for Cre concentrations of 6 uM and transduction times of 6 h. Longer incubation times do not significantly increase transduction efficiency. The optimal cell density for Cre protein transduction is about 50,000 hES cells per cm2 .

1. One day prior to protein transduction plate mitotically inactivated MEF cells on the desired number of tissue culture plates.

2. Remove the medium from the hES cells and add enough prewarmed accutase II solution to cover the colonies evenly. Incubate for 25 min at 37 oC.

3. Detach the cells with KO/SR medium and transfer them to a 50-ml centrifugation tube. Take a sample of 15 l and mix it with the equal volume of trypan blue solution to stain the cells. Take 15 l stained cells, count the cell number in a Fuchs-Rosenthal chamber, and calculate the total number of hES cells. Calculate how many cells are required for the desired number of tissue culture plates.

4. Transfer the calculated volume of the cell suspension to 15- or 50-ml centrifugation tubes and centrifuge for 5 min at 1000 × g and 4 oC.

5. Resuspend the pellet in KO/SR medium and dispense the cell suspension on the MEF cell-coated tissue culture plates. Allow the cells to adhere for 24 h at 37 oC and 5% CO2.

6. Dilute the Cre protein stock solution in KO/SR medium to the desired concentration.

7. Remove the KO/SR medium from the plated hES cells and add enough Cre-containing KO/SR medium to cover the cells evenly.

8. Incubate for 1–6 h at 37 oC and 5% CO2 .

9. Remove the Cre-containing medium, wash the cells once with knockout DMEM and add normal KO/SR medium again.

10. Prior further use of the Cre-transduced cells, allow the cells to recover for at least 24 h.