Abstract
Lysophosphatidic acid (LPA) is a lysophospholipid that acts as an extracellular signal through the activation of cognate G protein-coupled receptors (GPCRs). There are six known LPA receptors (LPA1–6). The first such receptor, LPA1, was identified in the embryonic brain and has been studied extensively for gene expression throughout the body, including through studies of receptor-null mice. However, identifying receptor protein expression in situ and in vivo within living cells and tissues has been difficult because of biologically low receptor expression and variable antibody specificity. To visualize native LPA1 receptor expression in situ, we generated a knock-in mouse produced by homologous recombination in murine embryonic stem (ES) cells to replace a wildtype Lpar1 allele with a mutant allele created by in-frame fusion of EGFP to the 4th exon of Lpar1 (Lpar1-EGFP knock-in allele). Homozygous knock-in mice appeared normal and the expected mendelian ratios of knock-in allele transmission were present in females and males. Histological assessments of the fetal and adult central nervous system (CNS) demonstrated expression patterns that were consistent with prior in situ hybridization studies. This new mouse line will be useful for studies of LPA1 in the developing and adult CNS, as well as other tissues, and for receptor assessments in living tissues and disease models.
Highlights
Molecular cloning of the first lysophospholipid receptor was reported 25 years ago from studies of the embryonic cerebral cortex [1], which identified a receptor known as LPA1 that mediates the effects of lysophosphatidic acid (LPA) [3,4,5]
Mouse Lpar1 genomic DNA fragments were amplified from a bacterial artificial chromosome (BAC) template containing the Lpar1 genomic locus (BAC RP23-149020, Children’s Hospital Oakland Research Institute) using Pfx50TM DNA polymerase (Invitrogen)
An amplified fragment containing Lpar1 exon 4 including a portion of the 3′ untranslated region (UTR) was used to fuse EGFP inframe to the carboxy terminus of Lpar1 using overlap PCR
Summary
Molecular cloning of the first lysophospholipid receptor was reported 25 years ago from studies of the embryonic cerebral cortex [1], which identified a receptor known as LPA1 (gene name Lpar for mouse, LPAR1 for human [2]) that mediates the effects of lysophosphatidic acid (LPA) [3,4,5]. While gene expression studies by in situ hybridization (ISH) identified Lpar expression in NPCs [1] during embryonic brain development, and oligodendrocytes [29] in the adult brain, determining the location of receptor protein has been more challenging because of inconsistent antibody specificity and availability, low receptor expression, differences in tissue preparation, and variables related to the effects of mouse background strain that produced inconsistent, if not contradictory results. We report an Lpar1-EGFP fusion knock-in transgenic mouse line and its initial characterization within the developing and adult brain
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