Targeting of the L-selectin gene to produce triple-null ES cells.

(A) Partial restriction map of the mouse L-selectin locus together with the targeting construct. Exons 2 to 6 are indicated as solid boxes. Most of the lectin domain (exon 3) is deleted and replaced with a puromycin resistance cassette in the orientation opposite to that of the L-selectin gene. The 5′, 3′, and internal probes used to detect the desired homologous recombination event are shown. (B) Schematic representation (not to scale) of the 3 selectin genes with the selectable markers inserted after the L-selectin gene was targeted incis to the original E- and P-selectin mutations. (C) Probing of Southern blots identified wild-type +/+, −/−, and +/− mouse genotypes for all 3 selectin mutations. The E-selectin genotype was identified by 4.3-kb mutant and 7.5-kb wild-type EcoRI fragments respectively10 and the P-selectin genotype by 13.8-kb wild-type and 3.9-kb mutant EcoRV fragments.15 The introduced L-selectin mutation was identified by a 7.2-kb wild-type XbaI fragment and a 4.4-kb mutant fragment. The blots show that the mutations segregate together, creating triple-selectin–null mice.