Generation of αL-I306A mice. (A) Structure of the mouse αL I domain built by homology modeling using a crystal structure of the human αL I domain (1ZON) as a template. The Mg2+ ion at the ligand binding site (ie, metal ion–dependent adhesion site) is shown with a green sphere and the side chain of I306 is in red. The amino (N) and the carboxyl (C) termini are labeled. Note that I306 is located in the hydrophobic pocket underneath the C-terminal helix. (B) Targeted insertion to the Itgal locus of the floxed ACN cassette and the mutated exon 9 (9*) that contains αL-I306A. The (1) targeting vector, (2) wild-type Itgal locus, (3) targeted Itgal allele containing floxed ACN cassette, and (4) mutated Itgal (I306A) allele are shown. Exons are shown as filled boxes. Long arm (LA) and short arm (SA) of homology, as well as the diphtheria toxin (DT) are shown. The floxed ACN cassette is deleted in chimeric male mice during spermatogenesis, leaving one loxP site (4). An engineered EcoRI site (E*) was designed to identify the targeted allele by Southern blot analysis. N indicates NcoI; E, EcoRI; A, AvrII; Sm, SmaI; and S, SpeI. The thick black line indicates the probe used to screen for homologous recombinations. (C) Genotyping and confirmation of deleted ACN cassette by polymerase chain reaction (PCR). Genomic DNA isolated from tails was used for PCR analyses. PCR bands are shown for wild-type (WT/WT, 300 bp), heterozygote (KI/WT, 300 and 390 bp), and homozygote (KI/KI, 390 bp) samples.
