A) Schematic representation of the hprt genomic locus and targeting vector with α-MHC-LacZ insert. The position of a Southern blot probe (probe b) containing LacZ specific sequences relative to Xba1 (x) and EcoRV (e) restriction enzyme cut sites is shown. B) Resistance to growth in HAT and sensitivity to G418 and 6-TG was restored in correctly targeted α MHC-LacZ ES cell lines. C) Southern blot analysis of genomic DNA from non-targeted F3 ES cells as well as from six targeted HAT-resistant ES cell lines generated from F3 cells is shown. The targeted allele is identified using probe b as a 7.0 kb EcoRV fragment. α MHC-LacZ ES cell line #4 was used to generate embryos shown in D. D) E8.5 embryos derived from α-MHC-LacZ targeted ES cells by tetraploid aggregation. β-galactosidase expression was identified by x-gal staining (blue). Expression was restricted to the developing heart (h).