DISRUPTION OF THE MURINE GENE ENCODING PHOSPHATIDYLETHANOLAMINE N-METHYLTRANSFERASE

All nucleated cells make phosphatidylcholine the CDP-choline pathway, Liver has an alternative pathway in which phosphatidylcholine is made by methylation of phosphatidylethanolamine catalyzed by phosphatidylet hanolamine N-methyltransferase (PEMT). We investigated the function of PEMT and its role in animal physiology by targeted disruption of its gene, Pempt2. A targeting vector that interrupts exon 2 was constructe d and introduced into mice yielding three genotypes: normal (+/+), het erozygotes (+/-), and homozygotes (-/-) for the disrupted PEMT gene, O nly a trace of PE methylation activity remained in Pempt2(-/-) mice, A ntibody to one form of the enzyme, PEMPT2, indicated complete loss of this protein from Pempt2(-/-) mice and a decrease in Pempt2(+/-) mice, compared with Pempt2(+/+) mice. The levels of hepatic phosphatidyleth anolamine and phosphatidylcholine were minimally affected, The active form of CTP:phosphocholine cytidylyltransferase, the regulated enzyme in the CDP-choline pathway, was increased 60% in the PEMT-deficient mi ce. Injection of [L-methyl-H-3] methionine demonstrated that the in vi vo PEMT activity was eliminated in the Pempt2(-/-) mice and markedly d ecreased in the Pempt2(+/-) mice, This experiment also demonstrated th at the choline moiety derived from PEMT in the liver can be distribute d via the plasma throughout the mouse where it is found as phosphatidy lcholine, lysophosphatidylcholine, and sphingomyelin. Mice homozygous for the disrupted Pempt2 gene displayed no abnormal phenotype, normal hepatocyte morphology, normal plasma lipid levels and no differences i n bile composition, This is the first application of the ''knockout mo use'' technique to a gene for phospholipid biosynthesis.