title: Construction of the subtracted cDNA library of striatal neurons treated with long-term morphine.
abstract: OBJECTIVE
  To construct a morphine tolerance model in primarily cultured striatal neurons, and screen the differentially expressed genes in this model using suppression subtractive hybridization (SSH).
  
  
  METHODS
  Sbtracted cDNA libraries were constructed using SSH from normal primarily cultured striatal neurons and long-term morphine treated striatal neurons (10-5 mol/L for 72 hours). To check reliability of the cell culture model, RT-PCR was performed to detect the cAMP-responsive element-binding protein (CREB) mRNA expression. The subtracted clones were prescreened by PCR. The clones containing inserted fragments from forward libraries were sequenced and submitted to GenBank for homology analysis. And the expression levels of genes of interest were confirmed by RT-PCR. Results CREB mRNA expression showed a significant increase in morphine treated striatal neurons (62.85 ± 1.98) compared with normal striatal neurons (28.43 ± 1.46, P < 0.01). Thirty-six clones containing inserted fragments were randomly chosen for sequence analysis. And the 36 clones showed homology with 19 known genes and 2 novel genes. The expression of 2 novel genes, mitochondrial carrier homolog 1 (Mtch1; 96.81 ± 2.04 vs. 44.20 ± 1.31, P < 0.01) and thymoma viral proto-oncogene 1 (Akt1; 122.10 ± 2.17 vs. 50.11 ± 2.01, P < 0.01), showed a significant increase in morphine-treated striatal neurons compared with normal striatal neurons.
  
  
  CONCLUSIONS
  A reliable differential cDNA library of striatal neurons treated with long-term morphine is constructed. Mtch1 and Akt1 might be the candidate genes for the development of morphine tolerance.
authors.affiliation: Department of Neurobiology, Jining Medical College, Jining 272067, China. bbai@mail.jnmc.edu.cn
authors.forename: Bo
authors.initials: B
authors.lastname: Bai
authors.forename: Hai-qing
authors.initials: HQ
authors.lastname: Liu
authors.forename: Jing
authors.initials: J
authors.lastname: Chen
authors.forename: Ya-lin
authors.initials: YL
authors.lastname: Li
authors.forename: Hui
authors.initials: H
authors.lastname: Du
authors.forename: Hai
authors.initials: H
authors.lastname: Lu
authors.forename: Peng-li
authors.initials: PL
authors.lastname: Yu
mesh_terms: D000701:Analgesics, Opioid / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D003342:Corpus Striatum / Q000166:cytology*; D004361:Drug Tolerance / Q000502:physiology; D020869:Gene Expression Profiling; D015723:Gene Library; D008969:Molecular Sequence Data; D009020:Morphine / Q000494:pharmacology*; D009474:Neurons / Q000166:cytology / Q000187:drug effects*; D009693:Nucleic Acid Hybridization / Q000379:methods; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
chemical_list: D000701:Analgesics, Opioid; D009020:Morphine