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Volumn 286, Issue 5449, 1999, Pages 2495-2498

Enhanced morphine analgesia in mice lacking β-arrestin 2

Author keywords

[No Author keywords available]

Indexed keywords

BETA ARRESTIN; G PROTEIN COUPLED RECEPTOR; GUANINE NUCLEOTIDE BINDING PROTEIN; MORPHINE;

EID: 0033601341     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.286.5449.2495     Document Type: Article
Times cited : (861)

References (44)
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    • A bacteriophage λ library of mouse 129SvJ genomic DNA (Stratagene) was screened with the rat βarr2 cDNA (24). Positive phages were identified and analyzed by restriction endonuclease digestion. A 12-kb βarr2 fragment was digested with Bam HI, subcloned into pBlue-script KS(-), and sequenced. The targeting vector was assembled by blunt-end ligation of a pHSV-TK cassette (from plasmid plC19R/MCl-TK), a 2.8-kb Nco I-Bam HI βarr2 fragment, a pGK-neo cassette (from plasmid pD383) that replaced the 0.8-kb Bam HI-Hind III fragment of βarr-2, and a 4.5-kb Hind III βarr2 fragment into pBluescript KS(-). This targeting vector was linearized with Not I and was electroporated into mouse embryonic stem cells. Genomic DNA from transfectants resistant to G418 and gancyclovir were isolated and screened by Southern blot analysis using a 0.2-kb 5′ external βarr2 probe and a 0.3-kb 3′ external βarr2 probe. Supplemental information regarding the targeting construct is available at Science Online (www.sciencemag.org/feature/data/1045481.shl). Chimeric animals were generated by microinjecting these embryonic stem cells into C57BL/6 blastocysts. Five chimeric male pups were obtained and mated with C57BL/6 females. Germ line transmission was confirmed by Southern blotting. Heterozygous offspring were intercrossed to obtain homozygous mice. Wild-type and mutant mice used in this study were age-matched, 3-to 5-month-old male siblings. For protein immunoblot analysis, whole-cell lysates were prepared by polytron homogenization in lysing buffer [10 mM tris (pH 7.4), 5 mM EDTA, one protease inhibitor tablet per 10 ml (Roche Molecular Biochemicals, Indianapolis, IN), and 1% Nonidet-40]. Polyacrylamide gels were loaded with 25 μg of protein per lane, and equivalent protein loading was confirmed by Ponceau S staining of the gels. After transfer to polyvinyldifluoride membranes, proteins were blotted with polydonal antibodies to β-arrestin 2 or β-arrestin 1 (24). Bands were visualized with secondary antibody conjugated to horseradish peroxidase and an enhanced chemiluminescence detection system (Amersham). All experiments were conducted in accordance with the NIH guidelines for the care and use of animals and with an approved animal protocol from the Duke University Animal Care and Use Committee.
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    • note
    • Mice were injected with morphine (10 mg/kg sc). After 30 min or 2 hours, wild-type mice were killed and blood was collected in vials containing sodium fluoride and potassium oxalate. Morphine concentrations in blood samples pooled from three mice per sample were 1500 ng/ml after 30 min and 83 ng/ml after 2 hours, as measured by mass spectroscopy analysis (Occupational Testing Division, LabCorp Inc., Research Triangle Park, NC). In similar experiments, βarr2-KO mice had a cocentration of 93 ng/ml in the blood after 2 hours.
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    • note
    • We thank B. H. Koller for homologous recombinant mouse embryonic stem cells and chimeric mice, M. R. Capecchi for plasmid plC19R/MCl-TK, and R. Hen for plasmid pD383. R.R.G. is a visiting scientist from the Institute of Pharmacology, Russian Academy of Medical Sciences, Moscow. Supported in part by NIH grants NS 19576 (M.G.C.) and HL16037, and by a cardiovascular unrestricted grant from Bristol-Myers Squibb (R.J.L). R.J.L. and M.G.C. are Investigators of the Howard Hughes Medical Institute.


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