Figure 1.

Targeted insertion of loxP sites into the Pig-a gene. (A) Part of the wild-type Pig-a locus showing the positions of exons 3–6, the targeting construct, and Pig-a allele containing the introduced loxP sites are shown. Solid and open boxes indicate the coding and noncoding exons, respectively. Restriction sites for BamHI (B) and EcoRI (E) are indicated. (B) Cre-mediated disappearance of GPI-anchored proteins on the surface of ES cells containing the loxP sites in the Pig-a gene. The plasmid pMC-Cre (13) with hyg^r was transfected into clone 58. The surface expression of heat-stable antigens (HSAg) was examined before and after selection with 150 μg/ml of hygromycin B for 7 days. Five of the 12 clones lost surface GPI-anchored proteins after selection. A representative clone that lost GPI-anchored proteins is shown. (C) Southern hybridization of the DNA from the germ-line-transmitted and control mice. Genomic DNA was obtained from tails and digested with BamHI. The 3.5- and 2.0-kb fragments represent targeted and endogenous alleles, respectively.