| Ornithine decarboxylase: Expression and regulation in rat brain and in transgenic mice | ||
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Mouse ODC cDNA was a kind gift from Dr. S. Gilmour (The Lankenau Medical Research Center, Philadelphia, PA, USA). The plasmid pODC1.7 was obtained by cloning a 1691 bp SalI - PvuII fragment of the mouse ODC cDNA into a pMSG eukaryotic expression vector (Pharmacia Biotech AB, Uppsala, Sweden) containing the dexamethasone-inducible mouse mammary tumor virus long terminal repeat (MMTVLTR) promoter. The plasmid pODC1.7(D233V) contained the SalI - PvuII fragment of the ODC cDNA in which the codon GAT, encoding the aspartate residue 233, was changed to GTT, encoding valine. Oligonucleotide-directed mutagenesis was carried out by subcloning the SalI - PvuII fragment into an M13 mp9 phage and using the “gapped duplex” method (Kramer et al. 1984) according to the manufacturer"s instructions (site directed mutagenesis kit, Boehringer Mannheim, Mannheim, Germany). The mutagenic oligonucleotide used was 5"-CTGCTTGTTATTGGT GG-3", mismatch being in italics. The sequences of DNA constructs were verified using the manual Sanger dideoxynucleotide method (Sanger et al. 1977).