Targeting of GFP to the MIXL1 locus in HESCs. (A) Structure of the gene targeting vector used to insert sequences encoding GFP into exon 1 of the MIXL1 locus using homologous recombination. PacI and NotI are restriction enzyme sites used to linearize the vector before electroporation. NeoR is the PGKNeo cassette encoding G418 resistance, flanked by loxP sites (black triangles). The positions of MfeI sites used to map the structure of the modified locus are shown, as are the position of primers (a, b) used to identify correctly targeted clones. (B) Southern blot analysis of MfeI digested genomic DNA shows that a 5′ external probe detects a fragment of 17 kb representing the endogenous locus from both parental HES3 cells and HESCs with a targeted MIXL1 locus. An additional fragment of 14.4 kb is also detected in the genetically modified cells, representing the distance from the 5′ external MfeI site to the 3′ end of GFP. (C) Probing this same DNA with GFP sequences indicates that these cells contain a single copy of the GFP gene consistent with a single genetic modification at the MIXL1 locus. (D) The integrity of sequences 3′ of the GFP gene was validated using a PCR-based approach (with primers a and b) to amplify DNA representing the junction of the targeting vector with the chromosomal DNA. Sequence analysis of this fragment confirmed that the relationship between the vector DNA and adjacent chromosomal sequences were as expected (data not shown).