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Volumn 292, Issue 5522, 2001, Pages 1728-1731

Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKBβ)

Author keywords

[No Author keywords available]

Indexed keywords

BLOOD; GENES; GLUCOSE; MUSCLE; PROTEINS;

EID: 0035368548     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.292.5522.1728     Document Type: Article
Times cited : (1601)

References (36)
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    • We employed mouse Akt2 specific PCR screening of 129/SvJ mouse genomic bacterial artificial chromosome (BAC) library (Genome Systems, St. Louis, MO) to clone the Akt2 gene. The BAC clone was digested with various restriction enzymes, shotgun subcloned into pBluescript (Stratagene, La Jolla, CA), and screened by hybridization with 32P-labeled human Akt2 cDNA. The subclones were used to map the mouse Akt2 locus. PCR-amplified fragments corresponding to recombination arms and floxed sequences were cloned into pFRTLOX (provided by M. A. Magnuson, Vanderbilt University School of Medicine), and linearized with Not I for electroporation into cultured E14 embryonic stem (ES) cells. After selection in G418 and ganciclovir, resistant clones were screened for homologous recombination by Southern blotting of Eco RI–digested genomic DNA and hybridization to Probe 1. Additional hybridization to a fragment within the floxed sequence was performed to ensure recombination upstream of the first LoxP site. To generate chimeric mice, we injected the targeted ES cells into C57Bl/6 blastocysts, which were subsequently implanted into pseudopregnant CD-1 foster mothers by the Transgenic and Chimeric Mouse Facility University of Pennsylvania. Resulting chimeric males were mated with C57BL/6 females to obtain agouti pups carrying the targeted allele, as determined by Southern blotting. To remove the floxed sequences, these mice were mated with Cre transgenic mice as described in the text. Transmission of the Cre transgene was followed by PCR specific for the transgene. In addition, hybridization of a probe corresponding to the intron between coding exons 4 and 5 was used to confirm the loss of the floxed region
    • 32P-labeled human Akt2 cDNA. The subclones were used to map the mouse Akt2 locus. PCR-amplified fragments corresponding to recombination arms and floxed sequences were cloned into pFRTLOX (provided by M. A. Magnuson, Vanderbilt University School of Medicine), and linearized with Not I for electroporation into cultured E14 embryonic stem (ES) cells. After selection in G418 and ganciclovir, resistant clones were screened for homologous recombination by Southern blotting of Eco RI–digested genomic DNA and hybridization to Probe 1. Additional hybridization to a fragment within the floxed sequence was performed to ensure recombination upstream of the first LoxP site. To generate chimeric mice, we injected the targeted ES cells into C57Bl/6 blastocysts, which were subsequently implanted into pseudopregnant CD-1 foster mothers by the Transgenic and Chimeric Mouse Facility at the University of Pennsylvania. Resulting chimeric males were mated with C57BL/6 females to obtain agouti pups carrying the targeted allele, as determined by Southern blotting. To remove the floxed sequences, these mice were mated with Cre transgenic mice as described in the text. Transmission of the Cre transgene was followed by PCR specific for the transgene. In addition, hybridization of a probe corresponding to the intron between coding exons 4 and 5 was used to confirm the loss of the floxed region.
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    • For Western blotting, liver, skeletal muscle, and isolated adipocytes or fat pads were homogenized in 50-mM tris buffer (pH 7.5), 1% NP-40, and protease inhibitor cocktail (Sigma, St. Louis, MO) with a Virtishear tissue disrupter. Protein concentration was determined by BCA Protein Assay Kit (Pierce, Rockford, IL) standardized with bovine serum albumin. Total lysate (50 μg) was resolved on SDS–7.5% polyacrylamide gel, and transferred to a nitrocelluose membrane. To detect Akt1, we used affinity-purified antibodies to the COOH-terminus of mouse Akt1, and to detect Glut4, we used affinity purified antibodies to the COOH-terminus of rat GLUT4 was used. Insulin receptor β-subunit was detected with rabbit polyclonal antibody (Santa Cruz, Santa Cruz, CA). Horseradish peroxidase-linked secondary antibody (Santa Cruz) was used for detection by enhanced chemiluminescence. For Northern blotting, 250 to 600 base pairs of 5'-proximal 3'-untranslated sequences to mouse Akt1, Akt2, or Akt3 were random-labeled. Full-length rat Glut4 cDNA was random-labeled for Glut4 expression analysis. Total RNA (10 μg) isolated with TRIZOL Reagent (Gibco-BRL, Grand Island, NY) or mRNA (2 μg) further purified with Oligotex mRNA Kit (Qiagen, Valencia, CA) was Northern blot analysis by standard techniques
    • For Western blotting, liver, skeletal muscle, and isolated adipocytes or fat pads were homogenized in 50-mM tris buffer (pH 7.5), 1% NP-40, and protease inhibitor cocktail (Sigma, St. Louis, MO) with a Virtishear tissue disrupter. Protein concentration was determined by BCA Protein Assay Kit (Pierce, Rockford, IL) standardized with bovine serum albumin. Total lysate (50 μg) was resolved on SDS–7.5% polyacrylamide gel, and transferred to a nitrocelluose membrane. To detect Akt1, we used affinity-purified antibodies to the COOH-terminus of mouse Akt1, and to detect Glut4, we used affinity purified antibodies to the COOH-terminus of rat GLUT4 was used. Insulin receptor β-subunit was detected with rabbit polyclonal antibody (Santa Cruz, Santa Cruz, CA). Horseradish peroxidase-linked secondary antibody (Santa Cruz) was used for detection by enhanced chemiluminescence. For Northern blotting, 250 to 600 base pairs of 5'-proximal 3'-untranslated sequences to mouse Akt1, Akt2, or Akt3 were random-labeled. Full-length rat Glut4 cDNA was random-labeled for Glut4 expression analysis. Total RNA (10 μg) isolated with TRIZOL Reagent (Gibco-BRL, Grand Island, NY) or mRNA (2 μg) further purified with Oligotex mRNA Kit (Qiagen, Valencia, CA) was submitted to Northern blot analysis by standard techniques.
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    • We thank J. Richa (Transgenic and Chimeric Mouse Facility, University of Pennsylvania) for the generation of chimeric mice and W. Ewens and B. Wolf (University of Pennsylvania) for advice on statistical analyses. The histological procedures were performed by the Center for Molecular Studies in Digestive and Liver Diseases (supported by NIH grant P30 DK50306). Serum insulin was assayed by the Radio-immunoassay Core Facility Penn Center for Diabetes (NIH grant P30 19525). H.C. is a recipient of the National Research Service Award for Training in Cell and Molecular Biology (GM07229). Supported in part by NIH grant RO1 DK56886 (to M.J.B
    • We thank J. Richa (Transgenic and Chimeric Mouse Facility, University of Pennsylvania) for the generation of chimeric mice and W. Ewens and B. Wolf (University of Pennsylvania) for advice on statistical analyses. The histological procedures were performed by the Center for Molecular Studies in Digestive and Liver Diseases (supported by NIH grant P30 DK50306). Serum insulin was assayed by the Radio-immunoassay Core Facility at the Penn Center for Diabetes (NIH grant P30 19525). H.C. is a recipient of the National Research Service Award for Training in Cell and Molecular Biology (GM07229). Supported in part by NIH grant RO1 DK56886 (to M.J.B.).


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