Fig. 2. Inactivation of LPP3 by homologous recombination in ES cells. (A) Gene targeting strategy. The exon containing the 3^rd outer loop, containing part of the catalytic domain (box), was deleted (top). The structure of the wild-type allele (middle) and targeted allele (bottom) after homologous recombination are shown. The fragments used for confirming 5' and 3' recombination as well as the location of the primers used for PCR genotyping (arrows) are indicated. (B) LPP3 genotyping by Southern blot. BamHI and BglI digested DNAs were tested with 5' and 3' probes respectively. (C) Genotype of embryos by PCR. DNA samples of yolk sacs were used for amplification of mutant and wild-type allele products using the set of 3 primers indicated in A. Wild-type product = 302 bp; mutant product = 500 bp. (D) Northern blot of embryoid body (EBs) total RNA shows the presence of a smaller transcript (-177 bp) in homozygous mutant cells resulting from the deletion of the exon. (E) Western blot of primary cultured cells revealed a reduction in the levels of LPP3 in heterozygous cells (compare to wild-type) and the absence of any intact protein in homozygous mutant cells. The 36 kDa upper band corresponds to the glycosylated form of the enzyme. (F) PKC phosphorylation in heterozygous and homozygous mutant EBs cultured for 12 days. A phospho (pan)-PKC antibody was used to indirectly measure PKC activation. A reduction of around 50% phospho-pan PKC was observed in the homozygous compared with the heterozygous tissues. Actin was used as a control for amount of protein loaded.