Figure 3

Genotype and G418 resistance of ES cells harboring insertions at the Hnf3α locus. a) A schematic showing the relative position of HindIII and EcoRV restriction endonuclease cut sites at the Hnf3α locus in ES cells. The predicted endonuclease fragment sizes identified by probe A (line) are shown above. Hnf3α ES cells have a wild type Hnf3α locus (Hnf3α, wt) and Hnf3αloxPNeo ES cells have a PgkloxP-Neo cassette introduced into a single Hnf3α allele. The pgk promoter was deleted from Hnf3αloxPNeo ES cells using Cre recombinase to generate G418 sensitive Hnf3αΔpgk-Neo ES cells. Hnf3αLacZ ES cells were generated by the introduction of a LacZ transgene into the Hnf3αΔpgk-Neo locus of Hnf3αΔpgk-Neo ES cells by homologous recombination. b) Southern blot of HindIII/EcoRV-digested genomic DNA isolated from Hnf3α, Hnf3αloxPNeo, Hnf3αΔpgk-Neo, and Hnf3α LacZ ES cells. Probe A identified an 8.0 kb wild type fragment in Hnf3α ES cells (lane 1). In Hnf3αloxPNeo (lane 2), Hnf3αΔpgk-Neo (lane 3), and Hnf3α LacZ (lane 4) ES cells probe A hybridized to an additional fragment of 4.0, 3.5 and 2.7 kb, respectively, due to the introduction of a novel EcoRV restriction endonuclease cut site. c) Hnf3α, Hnf3αloxPNeo, Hnf3αΔpgk-Neo, and Hnf3α LacZ ES cells were cultured in the absence (-G418) or presence (+G418) of 300 μg/ml of G418. Staining with methyl green identified G418-resistant ES cell colonies (blue dots).