Generation of HPRT negative ES cells. A) Schematic representation of the hprt genomic locus, targeting vector and mutant allele. The relative positions of exons (black boxes), BamH1 restriction endonuclease cut sites (b), and genes encoding neomycin phosphotransferase (neo) and HSV thymidine kinase (HSV-tk) selectable markers, as well as the position of the Southern blot probe (intron 3 probe) are shown. B) Growth of R1 hprt positive and F3 hprt negative ES cells in medium supplemented with G418, 6-TG, HAT or without drug (0 drug). Drug resistant ES cells are visible as blue colonies after staining with methyl green . C) Southern blot analysis of genomic DNA from R1 and F3 ES cells as well from the offspring of chimeric mice generated from F3 cells. The wild type hprt allele can be identified using intron 3 probe as a 7.0 kb BamH1 fragment and the targeted allele (mutant) as a 8.3 kb fragment.