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Fig. 1. Generation of PAPPA-null mice. (A) Schematic representation of the mouse gene in the region of exons 3 and 4 of the PAPPA locus, the replacement vector and the targeted allele. The position of the probe used for Southern analysis is indicated by the dark gray bar, and the sizes of the endogenous and targeted BamHI (B) genomic DNA fragments recognized by this probe are shown. An example of genotyping of mouse tail DNA is shown in the insert. Wild-type mice have a single 15 kb band (lanes 1, 8), heterozygous mice have both 15 kb and 2.6 kb bands (lanes 6, 7), and homozygous mutants have a single 2.6 kb band (lanes 2-5). (B) Weights of wild-type (+/+), heterozygous (+/-) and PAPPA-deficient (-/-) mice at birth. Results are mean±s.e.m.; n=20 for each genotype. ^*, significantly different from wild-type, P<0.0001. (C) Growth curves of wild-type (), heterozygous () and homozygous () PAPPA^-/- mutant mice. Values are mean±s.e.m. of 28-141 individual weights. (D) RT-PCR for PAPPA mRNA expression (upper panel) in tissues from wild-type (WT) and PAPPA^-/- mice. k, kidney; h, heart; li, liver; f, femur; t, tibia; b, brain; c, calvaria; lu, lung. -, negative control; +, positive control. Lower panel shows ethidium bromide staining of tissue 28S and 18S RNA to validate integrity and loading. (E) IGFBP4 proteolysis in medium conditioned by embryonic fibroblasts derived from wild-type (WT) and PAPPA^-/- mice. ¹²⁵I-IGFBP4 was incubated in MEF-conditioned medium without (-) or with (+) IGF2 for 6 hours. Reaction products were analyzed by SDS-PAGE and autoradiography. Arrows indicate intact IGFBP4 and 18 kD and 14 kD proteolytic fragments.

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