Disruption of the murine CTP: phosphoethanolamine cytidylyltransferase gene causes embryonic lethality

Abstract

The CDP: ethanolamine (Kennedy) pathway is responsible for the de novo biosynthesis of phosphatidylethanolamine (PE) and ethanolamine plasmalogens, where diacyl- and alkylacylglycerol are coupled with CDP-ethanolamine for their production respectively. We have disrupted the mouse gene encoding the rate-limiting enzyme in this pathway, CTP: phosphoethanolamine cytidylyltransferase (Pcyt2) by replacing a 2.8kb region, including the promoter region and exons 1–3 with a Neomycin cassette. Upon inter-crossing of Pcyt2+/- animals we obtained low litter sizes and unexpected Mendelian frequencies. From a total of 111 pups, 40% Pcyt2+/+ and 60% Pcyt2 +/− and no knockout pups were genotyped. Examination of embryos from E13.5 and E10.5 resulted in similar patterns of distribution, with no knockout embryos identified. This demonstrates that Pcyt2−/− mice are embryonic lethal prior to at least E10.5. Comparative mRNA analyses, immunoblotting and enzyme activity assays between Pcyt2+/− and controls have revealed a tissue specific gene dosage effect with significant differences between Pcyt2+/− and controls (30–50%). In vitro enzyme activities for PE methyltransferase, a liver specific enzyme that converts PE to phosphatidylcholine reveals slightly up-regulated activities in Pcyt2+/− animals. Phospholipid composition and content of Pcyt2+/− liver, heart and brain show no significant differences from controls. This demonstrates the importance of the Kennedy pathway for the production of ethanolamine phospholipids.