G1E cells are a GATA- (null) cell line derived from targeted disruption of GATA-1 in embryonic stem cells. These cells propagate in culture with a doubling time of about twelve hours. They resemble proerythroblasts and do not differentiate. They are dependent on Kit and Erythropoietin, but not IL-3. In our studies of erythroid differentiation, we have employed a G1E subclone designated G1E-ER4 which stably expresses a fusion product combining GATA-1 with the estradiol receptor ligand binding domain. Addition of either estradiol or taxoxifen (4-OHT) to the medium results in the functional activation of GATA-1. This triggers erythroid differentiation which becomes morphologically apparent at about 12 hours. By twenty-four hours, hemoglobin can be detected by benzidine staining. Microarray analysis indicates that transcriptional changes take place within the first hour of GATA-1 activation, however. By 30 hours, G1E cells reach a stage developmentally equivalent to an orthrochromatic erythroblast. They continue to hemoglobinize up to 72 hours, but then undergo apoptosis. They do not extrude their nuclei. Cells remain in log phase when maintained between 1 x 105 and 1 x 106 cells/mL; effectively this means passaging daily with splits between 1:4 and 1:10.
Puromycin is maintained as 1000X stock at -20C and can be added directly to culture flasks to maintain selection pressure on GIE-ER4. When a new batch is thawed, it is routine passed for one or two generations in media containing puromycin to select against revertants lacking GATA-1-ER.
Benzidine Staining (to assess hemoglobin content)
Dissolve 60 mg of o-diansidine (Sigma D-9143) in 29.7 mL H2O and 0.5 mL glacial acetic acid. Note o-diansidine is a toxic carcinogen; wear gloves and work in fume hood. To dissolve, heat gently and stir for 30-60 minutes. Centrifuge to eliminate undissolved particulate matter. Store supernatant at 4C in light-proof container. Reagent is good for several months, but should be discarded when it turns brown.
Prior to staining, prepare fresh benzidine reagent: one part hydrogen peroxide to 10 parts benzidine reagent.
Quantitation of cells in suspension
To create slides
May-Grunwald/Giemsa Stain (for morphology)
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