Mecr^{tm1.1(fabI)Akast}

A sequence encoding the bacterial fabI ETR (2-enoyl-thioester reductase), a functional prokaryotic ortholog of Mecr, was introduced into exon 2 of the mouse Mecr gene. The construct was C-terminally tagged with a double HA epitope tag. The splicing acceptor sites of exon 2 and 54 nucleotides of the exon 2 itself were maintained in-frame with the fabI coding region. The Mecr mitochondrial targeting sequence (MTS) is encoded by exon 1. These alterations are predicted to yield a chimeric mRNA encoding HA-tagged fabI N-terminally extended with the Mecr MTS. The construct was followed by the strong transcriptional terminator BGH attached to an FRT-flanked selection cassette to exclude the possibility of producing intact Mecr. A Mecrtm(fabl) mouse line devoid of the selection cassette was generated by deleting the FRT-flanked selection cassette by breeding with Tg(ACTFLPe)9205Dym/J mice. RT-PCR analysis revealed robust fabI mRNA transcript in E9.5 homozygous mutant embryos whereas fabI mRNA levels were ~25% in heterozygous embryos and essentially absent in wild-type embryos. Mecr mRNA expression was reduced to background levels and Western blot analysis confirmed the absence of Mecr protein expression in homozygous mutant embryos. A faint lipoylated Dlat signal was observed in protein extracts from homozygous embryos at E9.5, indicating a mild rescue of the enoyl-reductase deficiency. (J:242847)