FIG.1.

Targeted mutation of the mouse CD148 gene. (A) Schematic diagram (from top to bottom) of the wild-type CD148 allele encoding the cytoplasmic domain, the targeting vector, and the recombinant mutant allele, CD148^ΔCyGFP. Exons are indicated by vertical black rectangles. The phosphatase domain was disrupted by an in-frame insertion of the enhanced GFP coding sequence, as described in Materials and Methods. The loxP (black triangle)-flanked neomycin cassette (neo) permitted positive selection and subsequent excision by Cre recombinase, while the diphtheria toxin A cassette, DT-A, negatively selected nonhomologous recombinants. Xh, XhoI; RI, EcoRI; K, KpnI; P, PstI; Sc, ScaI; HIII, HindIII; S, SalI. (B) Southern blot analysis of E9.5 embryos from CD148^+/ΔCyGFP heterozygote intercrosses. Using the 5′ (10.0 kb and 2.5 kb) and 3′ (10.0 kb and 8.5 kb) probes as indicated in panel A, EcoRΙ restriction fragments were used to distinguish wild-type from mutant alleles. The asterisk indicates the presence of a pseudogene. (C) RT-PCR analysis of E9.5 yolk sac mRNA. Amplified products, determined using the primer set described in Materials and Methods, were 104 bp (wild type) and 159 bp (mutant), as predicted. (Right panel) Northern blot analysis of total RNA from adult kidneys. Total RNA was isolated, transferred, and hybridized with GFP cDNA probe (see Materials and Methods), using RNA from a GFP transgenic mouse (GFP TG) (20) as a positive control. The predicted 5.2-kb CD148^ΔCyGFP transcript (arrow) was detected in heterozygous mice. (D) Western blot analysis of adult mouse kidneys. Expressed CD148 forms were recovered from adult kidney extracts by a lectin (wheat germ agglutinin [WGA] from Triticum vulgaris) affinity step (left panel) or by GFP antibodies (right panel) and detected on immunoblots with an ectodomain interactive anti-CD148 or anti-GFP antibody. A doublet (brace) expressed from the mutant allele at approximately 200 kDa that demonstrates similar glycosylation heterogeneity to that of the native protein is recovered by lectin and by anti-GFP at a mass similar to that of the wild-type protein. The CHO cell lysates transfected with empty vector (pSRα) and CD148^ΔCyGFP expression plasmid (pSRα ΔCyGFP) were used as controls. Immunodepletion experiments using anti-GFP showed an approximately 30% reduction of CD148 compared with those using a control (bottom panel). (E) Photomicrographs of wild-type and CD148^ΔCyGFP homozygous embryos at E9.5 to 10.5. Homozygous mutant embryo at E9.5 shows reduced size, edema, and distended pericardium (arrowhead). At E10.5, severe growth retardation and prominent extension of pericardial sac (arrow) were noted.