The objective of this new initiative is to clone the dopamine and other monoamine transporters from monkey brain and develop stable cell lines that express non-human primate dopamine transporter cDNA's The monkey dopamine transporter coding sequences will provide information on the evolution of this critical brain transport system from monkey to human The cell lines expressing the monkey dopamine transporter clones will be used to screen novel drugs with diagnostic and therapeutic potential It will obviate the need to depend on availability of monkey brain tissue, reduce the hazard associated with fresh tissue, and provide consistency Finally, a cell line expressing the transporter will enable investigation of transporter regulation We have cloned dopamine transporter cDNAs from rhesus, cynomolgus and squirrel monkeys using a reverse transcriptase-polymerase chain reaction strategy, based on the high degree of similarity between the cloned mammalian dopamine transporte r sequences RNA was extracted from the substantia nigra and reverse transcribed into cDNA Oligonucleotides corresponding to highly conserved regions that are 5' and 3' to the dopamine transporter coding region were used to amplify the dopamine transporter coding region PCR products of the correct size were directly ligated into the expression vector pcDNA3 1/V5/His-TOPO and plus and minus strands were fully sequenced We are now expressing these monkey dopamine transporter expression constructs in HEK-293 cells The data demonstrate that 1 All monkey dopamine transporter coding regions differ from the human sequence; 2 At the protein level, all the monkey sequences contain a consensus cluster of amino acids in the second extracellular loop which differ from the human transporter; 3 Other differences are limited to single amino acid changes, none in transmembrane-spanning domains PUBLICATION Miller GM, Sajdel-Sulkowska EM, Madras BK Cloning of dopamine transporter coding regions from new and old world monkey Soc Neurosci Abst 24 609 1998
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