FIG. 3.

Targeted disruption of the mNle gene. (A) Structure of the WT mNle allele, the targeting vector, and the targeted allele. Exons (solid boxes), LoxP sequences (triangles), and positions of primers used for genotype analysis (a to f, bars), probes used for Southern blot analysis (bars), and pgk-DTA and pgk-Neo cassettes used for negative and positive selection are indicated. Restriction sites relevant to the targeting construct and to the screening strategies are as follows: BglI (Bg), BsrGI (Bs), EcoRV (E), HincII (H), and PstI (P). (B) Southern blot analysis of genomic DNA obtained from wild-type and heterozygous ES cells. (C) Genotype analysis of early embryos from mNle^+/− intercrosses. The first round of PCR used primers b, c, and d. The second round of PCR used primers a, e, and f, yielding amplification products of 159 bp and 210 bp for the wild-type and mutant alleles, respectively.