TITLE: ASSESSING RTTA ACTIVITY (Support Protocol 6) 
AUTHORS: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward

[STEPS]
?. Prepare four wells of the cell line of interest by EDTA split (Basic Protocol 1).
For at least the first time performing this protocol, include positive and negative control cells in order to compare with cells of interest.
?. 1 to 2 days after plating, follow Basic Protocol 2 (steps 6 to 9) for two of the wells in order to transfect with the pBI-MCS-EGFP plasmid.
?. One day later, aspirate medium and replace with fresh E8. In one trans- fected well and one un-transfected well, supplement medium with 2 μg/ml doxycycline.
If cells are more than 30% confluent when transfected, doxycycline may be added at the time of transfection in order to permit imaging the next day.
?. One day later, aspirate medium, rinse twice with PBS, and image with a fluorescent microscope.
eGFP typically has a half-life of 24 hr, so fluorescence should persist for at least 2 to 3 days after doxycycline treatment.
?. Since transfection is heterogeneous, only a subset of cells should express GFP. In addition, levels of expression may vary by the number of plasmid copies delivered to each cell. However, in cells with active tetracycline transactivator, the average intensity of GFP will be notably higher in the well exposed to doxycycline than in the well which was not. Some leaky expression is expected due to the sequence of the tetracycline operator in this plasmid, so un-transfected cells thus serve as a further negative control.
?. For each transfection, add  of Opti-MEM and  of total DNA to one 1.5-ml microcentrifuge tube and vortex for  to . In a second tube, combine  of Opti-MEM and  of Lipofectamine Stem reagent, and vortex for  to .
100 µl
3 µg
100 µl
10 µl
For TALEN-mediated insertion to the AAVS1 or CLYBL locus, such as for the hNGN2 (Addgene, cat. no. 105840) and hNIL (Addgene. cat. no. 105841) differentiation cassettes, use a 2:1:1 ratio of   donor construct with   of each of the site-specific TALENs. For AAVS1:   of pTALdNC-AAVS1_T2 (Addgene, cat. no. 80496) and   of pTALdNC-AAVS1_T1 (Addgene, cat. no. 80495) per transfection. For CLYBL:   of pZT-C13-R1 (Addgene, cat. no. 62197) and   of pZT-C13-L1 (Addgene, cat. no. 62196) per transfection.
?. Combine the contents of the two tubes and vortex again for  to . Incubate thismixture for  at .
0 Room temperature
?. Retrieve the cells plated in steps 3 to 4, and, using a P200 pipet tip and repeat pipettor, add  of the complete transfection solution from step 6 dropwise, evenly across the surface of the well. Return the cells to the incubator overnight.
200 µl
?. after transfection, aspirate transfection medium and replace with fresh E8. If applicable, evaluate transfection efficiency by fluorescence microscopy.
All cells transfected with the hNGN2 (Addgene, cat. no. 105840 or 110492) and hNIL constructs (Addgene, cat. no. 105841 or 105842) will transiently express mCherry for 3 to 4 days, while only those cells with transgene insertion will maintain stable expression of mCherry for longer periods of time. See Basic Protocols 3 and 4 for options for enrichment and clonal isolation.