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

=== Cre Recombinase Transduction into hES Cell-Derived Neural Precursors ===

Prepare neural precursor cells from hES cells according to a protocol published by Zhang et al. (In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat. Biotechnol. 2001 19, 1129–1133).

Transfer embryoid bodies (EBs) to PO-coated cell culture plates.

Propagate cells for 10 days in ITSFn medium. Within this time period, neural tube-like structures develop in the EB. These structures are isolated mechanically and are propagated as free-floating neurospheres in N2 medium for 2 weeks. Finally, isolate neural precursor cells by enzymatic dissociation of these neurospheres, plate on PO/laminin-coated culture plates and further cultivate in the presence of FGF2 according to Zhang's protocol. Alternatively, human neural precursors may be derived from fetal Central Nervous System (CNS) tissue based on Brüstle's protocol (1998, Chimeric brains generated by intraventricular transplantation of fetal human brain cells into embryonic rats. Nat. Biotechnol. 16,1040–1044). Optimal parameters for Cre transduction in ES cell-derived neural precursors are a Cre concentration of 1 uM and a transduction time of 6 h. For maximum survival, it is crucial that the cells are confluent before transduction.

1. Dilute the Cre protein stock solution in the medium used for the cultivation of neural precursors to a final concentration of 1 uM.

2. Remove the culture medium from the neural precursor cells and add the Cre protein solution. Incubate for 6 h at 37 oC and 5% CO2.

3. Wash the cells twice with DMEM&nbsp;: F12 medium, then add again the suitable culture medium.

4. Before further use, allow the cells to recover for at least 24 h.