Figure 4. Generation of γ1 EQ conditional knock-in mice.

(A) Schematic representation of the generation of Lamc1^cEQ. The open boxes represent exons. The protein coding sequences are indicated in gray. The targeting construct was designed to replace WT exon 28 encoding Glu at residue 1,605 with a floxed exon 28 followed by a mutated exon 28 encoding Gln at residue 1,605. The probes used for Southern blotting are indicated by bold lines. (B) Southern blot analyses of genomic DNA from WT, Lamc1^cEQ(neo)/+, and Lamc1^cEQ/+ offspring after digestion with SexAI and PacI. The detection of 9.0 and 7.4 kbp fragments with probe 1 and probe 2, respectively, in the Lamc1^cEQ(neo)/+ lanes indicates occurrence of the expected homologous recombination. The detection of a 7.4 kbp fragment with probe 1 in the Lamc1^cEQ/+ lane indicates that the neomycin-resistance gene has been removed from the Lamc1^cEQ(neo) allele by the Cre-loxP system. (C) Genotypes of offspring obtained from Lamc1^cEQ/+ intercrosses. (D–I) Histochemical analyses of Lamc1^cEQ/cEQ (D–F) and Tie2-cre;Lamc1^cEQ/cEQ (G–I) retinas. (D, G) In situ binding of recombinant integrin α3β1 (magenta) to frozen retinal sections. (E, H) Counterstaining of vascular BM with an anti–laminin-α5 antibody (green). Merged images with nuclear staining (blue) are also shown (F and I). Retinal vasculatures are indicated by filled (Lamc1^cEQ/cEQ) and open (Tie2-cre;Lamc1^cEQ/cEQ) arrowheads. Bars, 50 μm. (J) Genotypes of offspring obtained from mating between Lamc1^cEQ/+ female and Tie2-cre;Lamc1^cEQ/+ male mice. Only Lamc1^cEQ/cEQ and Tie2-cre;Lamc1^cEQ/cEQ mice are shown. S, SexAI restriction site; P, PacI restriction site; TK, thymidine kinase gene.