Generation of LPA₄-deficient mice. (A) Strategy for homologous recombination at the mouse Lpa4 gene locus on the X chromosome. The entire ORF of LPA₄ was replaced by the LacZ-neo cassette. The structure of the intact Lpa4 allele is shown at the top, the structure of the Lpa4 targeting vector is depicted in the middle, and the predicted structure of the targeted allele is shown at the bottom. (B) Southern blot analysis of genomic DNA from ES cells. Eco81I-digested genomic DNA samples from WT and LPA₄-deficient ES cells were hybridized with the 5′ probe shown in panel A. Lane 1 is WT, while lanes 2, 3, and 4 represent LPA₄-deficient cells. (C) Southern blot analysis of genomic DNA from ES cells. TaqI-digested genomic DNA samples from WT and LPA₄-deficient ES cells were hybridized with the 3′ probe shown in panel A. Lane 1 is WT, while lanes 2, 3, and 4 represent LPA₄-deficient cells. (D) PCR genotyping with genomic DNA from female tails. WT, wild-type; HT, heterozygous; KO, LPA₄-deficient. (E) Northern blot analysis of Lpa4 mRNA expression. Each of total RNA samples (5 μg) from E12.5 whole embryos were hybridized with a probe containing the entire ORF of Lpa4 or a human β-actin cDNA probe. (F) RT-PCR with RNA samples from LPA₄-deficient and WT mice. RNA was extracted from E12.5 whole embryos.