Construction of MIDGE vectors. (A) POMC exon 2-3 cDNA, encoding the signal peptide, adrenocorticotropic hormone (ACTH), melanocyte-stimulating hormone (MSH) and β-endorphin (END), was spliced into the pMOK plasmid between the SacI and KpnI restriction sites. The pMOK plasmid contains a kanamycin resistance gene, a cytomegalovirus promoter (PCMV), a chimeric intron and simian virus (SV) 40 polyadenylation (pA) site. The POMC-MIDGE-NLS was derived from the pMOK-POMC plasmid by cutting it with Eco31I, ligation with hairpin ODNs at both ends, and coupling of nuclear localization sequence (NLS) to one of the hairpin ODN. The other vectors were prepared analogously by modifying POMC gene as follows: (B) END sequence was removed to obtain control MIDGE-NLS; (C) ACTH and MSH fragments were removed while END sequence was preserved in its original place to obtain 1xEND-MIDGE-NLS encoding 1 copy of END; (D) ACTH fragment was removed, original END sequence was preserved and MSH fragment was replaced with an additional sequence of END to obtain 2xEND-MIDGE-NLS encoding 2 copies of END; (E) original END sequence was preserved and the ACTH and MSH fragments were replaced with additional END sequences to obtain 3xEND-MIDGE-NLS encoding 3 copies of END.