A transcriptional control system causes FingRs to be expressed at the same level as the endogenous target protein. (A) GPHN.FingR-GFP (green) expressed for 7 days localizes in a nonspecific pattern, likely due to high background from unbound FingR. (B, C) Endogenous Gephyrin (red) does not colocalize with GPHN.FingR-GFP. (D) GPHN.FingR-GFP (green) with transcriptional regulation expressed for 7 days appears in a punctate pattern that precisely colocalizes with that of endogenous Gephyrin (red, E, F). Note that red staining that does not colocalize in (F) is from untransfected cells. Scale bar is 5 μm. (G) To control its transcription the FingR (blue) is fused to a transcription factor consisting of a Zinc-Finger DNA binding domain (ZF, purple) fused with a KRAB(A) transcriptional repressor domain (pink). In addition, a ZF DNA binding site (light blue) was inserted upstream of the CMV promoter. When less than 100% of the target is bound by FingR, then 100% of the FingR binds to endogenous target and is prevented from moving to the nucleus. When 100% of the target is bound, then the FingR can no longer bind to the target, and, instead, moves to the nucleus as a result of the nuclear localization signal within the ZF. Once in the nucleus the transcription factor binds to the ZF binding site and represses transcription. Thus, the level of FingR is matched to the level of the endogenous target protein. (H) Live cortical neuron co-expressing regulated PSD95.FingR-GFP (green) and GPHN.FingR-mKate2 (red). Note that the regulation systems for PSD95.FingR-GFP and GPHN.FingR-mKate2 are based on DNA binding domains from CCR5 and IL2RG, respectively. Each FingR is expressed in a punctate, nonoverlapping fashion consistent with labeling of PSD-95 and Gephyrin. (I) Neuron in cortical culture expressing transcriptionally controlled GPHN.FingR-GFP following lentiviral infection. Images taken at 1 second intervals show a vesicle moving with a velocity of ~7 μm.s−1 in the axon. (J) Similarly labeled vesicle moving at ~4 μm.s−1 in the axon. Scale bar is 5 μm. See also , .