Hypothalami were dissociated with trypsin and viability monitored by trypan blue exclusion. Cells were plated onto poly D lysine pre coated 60 mm Petri dishes in DMEM supplemented with 10% fetal bovine serum, 0. 25% glucose, 2 mM glutamine, 3. 3 mg ml insulin, 1% antibiotic antimycotic and 1% vitamin solution. Cultures were maintained in a REVCO incubator at 37 C in humi dified air 7% CO2. Twenty four hours after seeding, cells were transfected essentially as described. In general, 8 mg of branched polyethylenimine solution was diluted in 10 ml of water, pH adjusted to 6. 9 with 0. 2 N HCl and the solution filtered. PEI and plasmid DNA were separately diluted to adjust NaCl to 150 mM in a final volume of 50 ul, vor texed and incubated for 10 min at room temperature, subsequently, the polymer solution was added to the DNA, vortexed mixed, incubated for 10 min at room temperature followed by the addition of 900 ul of serum free DMEM.

The supplemented DMEM was removed Inhibitors,Modulators,Libraries from the culture dishes and the transfection mixture was added. Inhibitors,Modulators,Libraries After 3 hours incubation, transfection mix was removed and fresh supplemented DMEM was added. Forty eight hours after transfection, cells were trypsinized and AV-951 subjected to FACS. Plasmid construct The minimal Trh promoter conferring tissue specific expression was excised with EcoR1 and BamH1 EcoRV digestion from the pNASS rTRH Luc expression vector. The Trh promoter fragment sticky ends were filled with Klenow DNA polymerase and subsequently sub cloned into the SacI BamH1 sites present on the pACT2 vector.

Finally, the Trh promoter fragment was cloned into the SacI BamHI sites in the phrGFP promoterless expression vector and Fluorescence activated cell sorting For preparative cell sorting, 5 Inhibitors,Modulators,Libraries �� 106 hypothalamic cells plated on 60 mm dishes were transfected as described Inhibitors,Modulators,Libraries above. After 48 h, cells were trypsinized, washed, resus pended in PBS 1% FBS and filtered through a 40 um nylon mesh. Cells were purified from a pool of five 60 mm dishes using the FACS Vantage and the exclusion method at high speed. Cells were sorted using the settings previously described and analyzed by analytical flow cytometry as described below. In general, 20,000 GFP cells were puri fied from 5 �� 106 cells. The percentage of GFP cells before and after purifica tion by preparative cell sorting was determined by analy tical flow cytometry using the FACS Vantage. All data acquisition and analyses were performed using the Cell Quest software. To estimate the number of GFP cells, a FL1 histogram was generated and positive cells were defined as those cells in the region M1. The percentage of cells in M1 from the empty vector transfected cells was subtracted from the percen tage of plasmid transfected cells in M1.