Fig. 1. Generation and analysis of ES cells and mice lacking the nidogen-binding module 1III4. (A) Restriction map for the genomic region of domain III of the Lamc1 gene. The sequence coding for the nidogen-binding module 1III4 is depicted in gray. The targeting vector and the resulting targeted allele after homologous recombination in ES cells are shown below. The final targeted allele was achieved by Cre-mediated deletion of the selection cassette. Predicted sizes of genomic fragments after EcoRV restriction and Southern blotting, using the externally located 5' probe, are indicated. A, ApaI; B, BamHI; EV, EcoRV; H, HindIII; Nh, NheI. (B) Southern blot analysis of EcoRV digests of genomic DNA from respective ES cell clones (left panel) and F₂ embryos at 18.5 dpc (right panel). A fragment of 7.5 kb is detected in the wild type. In addition, a 4.5 kb fragment is seen in heterozygous cells, which is further reduced in size to 2.7 kb after transient transfection with Cre (+/–^cre). Homozygous mutant embryos were identified by the appearance of a single 2.7 kb band (right panel), whereas homozygous ES cells showed both a 4.5 and a 2.7 kb band (left panel). (C) RT-PCR analysis of RNA isolated from wild-type (+/+), heterozygous (+/–) and homozygous mutant (–/–) ES cells. Primer pairs spanning the deletion site resulted in the expected size of fragments for the wild-type (369 bp) and mutated (201 bp) laminin 1 chain (top panel). In all genotypes, the downstream located modules 1III6-7 (middle panel) are normally transcribed.