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Birnbaum Laboratory

Summary:

The ability to respond to nutritional stress is one of the most primitive adaptations that organism must accomplish. The pathways that alert the organism to an absence of food and initiate an appropriate response are remarkably well-conserved and involve such critical signaling molecules as the protein kinases Akt and AMP-activated protein kinase (AMPK) as well as nutrient sensors such as the carbohydrate response element binding protein (ChREBP).

The Birnbaum lab studies this complex biological response in two contexts: the initiation of cell growth after a transition from nutritional deprivation to abundance and the insulin-dependent redistribution of simple substrates into long-term energy stores. The latter process involves a number of distinct but interacting components such as glucose-stimulated insulin secretion, and the insulin-dependent acceleration of hepatic lipid synthesis and glucose uptake into adipocytes and muscle. Two aspects of the regulation of glucose transport by insulin, both of which are studied in the Birnbaum lab, are the way in which insulin regulates the movement of hormone-sensitive Glut4 glucose transporter from the inside of the cell to the plasma membrane, and the signaling pathway by which insulin accomplishes this. There are also a number of projects underway aimed at understanding how the evolutionarily conserved sensor of nutritional stress, AMP-activated protein kinase, regulates carbohydrate and fat metabolism. These fundamental biological problems are addressed using experiments performed in tissue culture cells, mice and the genetically tractable organism Drosophila melanogaster.

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Organisms and Viruses

  • Akt1loxP/loxP ( Mus musculus )

    J1 embryonic stem cells were electroporated with NotI-digested Akt1 targeting vector and subjected to selection with G418, and the clones were screened for homologous recombination by PCR amplification of the intron 4 region and Southern blot analysis. Cells that were heterozygous for loxP sites flanking exons 3 and 4 of the Akt1 gene were injected into blastocysts derived from C57BL/6J mice and implanted into pseudopregnant CD-1 foster mothers according to standard methods. Chimeric mice (>90% as judged by coat color) were then bred with C57BL/6J mice.

    This mouse strain can be used to generate tissue-specific Akt1 deletions.

  • Akt1loxP/loxP;Akt2loxP/loxP ( Mus musculus )

    This strain can be used to develop tissue-specific Akt1 and Akt2 knockout mice.

  • Akt1loxP/loxP;Akt2loxP/loxP;Foxo1loxP/loxP ( Mus musculus )

    These mice can be used to generate tissue-specific knockouts of Akt1, Akt2 and Foxo1.

  • Akt1myr (RIPp) ( Mus musculus )

    Rat insulin II promoter drives expression in pancreatic beta cells of a form of Akt1 rendered constitutively active by the addition of an N-terminal myristoylation sequence (myr-Akt1).

    The transgene product is smaller than the endogenous Akt1 due to the mutant protein's deletion of its pleckstrin homology domain. In the pancreas, the transgene was expressed exclusively in the endocrine cells as assessed by immunohistochemistry using an antibody against the epitope tag.

  • Akt2fl/fl ( Mus musculus )

    "An frt-flanked PGK-neomycin resistance cassette was inserted into intron 5, and loxP sites were inserted flanking exons 4 and 5."

  • Akt2loxP/loxP ( Mus musculus )

    Akt2 exons 3 and 4 are flanked by loxP sites. This strain can be used to develop tissue-specific Akt2 knockout mice.

  • B6.129P2-Akt1 tm1Mbb/J ( Mus musculus )

    "Mice that are homozygous for the targeted mutation are viable and do not display any gross behavioral abnormalities. Homozygotes exhibit lower fertility. Female homozygotes do not nurse well; up to 50% perinatal mortality can occur. No gene product (mRNA or protein) is detected by Northern or Western blot analysis of mouse embryonic fibroblasts. Homozygotes are only 80% of wildtype body weight at birth, and remain small. This mutant mouse strain may be useful in related to organismal growth."

  • B6.Cg-Akt2 tm1.1Mbb/J ( Mus musculus )

    Mice with a targeted disruption in the Akt2 locus were created by homologous recombination. The targeting vector was designed to insert LoxP sites flanking the sequence containing the coding exons 4 and 5. Mice harboring the targeted allele were identified by Southern blotting and were mated to transgenic mice expressing Cre recombinase driven by a 6-kb 5′-flanking sequence from the Brn/Pou3f4 gene to cause germ-line excision of exons 4 and 5. Exon 5 encodes the lysine residue necessary for catalytic activity. Cre recombinase-mediated deletion of exons 4 and 5 results in a frameshift mutation leading to a premature termination even if the remaining exon 3 were to splice to exon 6. The progeny carrying both the Cre transgene and the targeted allele were mated with wild-type (WT) mice to obtain offspring in which the Cre transgene was segregated away and the targeted allele was excised, as determined by the polymerase chain reaction (PCR) and Southern blotting, respectively. These mice were mated inter se to produce offspring with homozygous deletions of Akt2.

  • B6.Cg-Akt2(tm1.1Mbb/J) ( Mus musculus )

    "Mice were generated with a targeted disruption in the Akt2 locus by homologous recombination. The targeting vector was designed to insert LoxP sites flanking the sequence containing the coding exons 4 and 5 Mice harboring the targeted allele were identified by Southern blotting and were mated to transgenic mice expressing Cre recombinase.
    The progeny carrying both the Cre transgene and the targeted allele were mated with wild-type (WT) mice to obtain offspring in which the Cre transgene was segregated away and the targeted allele was excised, as determined by the polymerase chain reaction (PCR) and Southern blotting, respectively. These mice were mated inter se to produce offspring with homozygous deletions of Akt2."

  • Liver-specific Akt1 and Akt2 knockout ( Mus musculus )

    The liver-specific Akt1;Akt2 KO mice were created by injecting Akt1loxP/loxP;Akt2 loxP/loxP mice with adeno-associated virus expressing Cre recombinase under the control of the Tbg promoter (AAV-Tbg-Cre).

  • Liver-specific Akt2 KO ( Mus musculus )

    Liver-specific Akt2 KO mice were created by injecting Akt2 loxP/loxP mice with adeno-associated virus expressing Cre recombinase under the control of the Tbg promoter (AAV-Tbg-Cre).

  • Liver-specific knockout of Akt1, Akt2, and Foxo1 ( Mus musculus )

    Akt1loxP/loxP;Akt2loxP/loxP mice were crossed with Foxo1loxP/loxP mice. The liver-specific Akt1;Akt2;Foxo1 knockout mice were created by injecting Akt1loxP/loxP;Akt2 loxP/loxP;Foxo1loxP/loxP mice with adeno-associated virus expressing Cre recombinase under the control of the Tbg promoter (AAV-Tbg-Cre).

  • Systemic Akt2 KO and liver-specific Akt1 KO ( Mus musculus )

    Akt1loxP/loxP;Akt2−/− mice were bred to Afp–Cre mice. Afp-Cre mice express Cre recombinase specifically in the liver.

  • UAS-dAMPKalpha K56R.B13 ( Drosophila melanogaster )

    Kinase-dead K56R mutation was introduced by site-directed mutagenesis. Constructs were injected into Drosophila embryos at Duke University Model Systems Genomics (Durham, NC).

  • UAS-dAMPKalpha WT.B32 ( Drosophila melanogaster )

    Gateway cloning (Invitrogen) was used to generate pUAST-dAMPKalpha. Constructs were injected into Drosophila embryos at Duke University Model Systems Genomics (Durham, NC).

  • UAS-myrAkt ( Drosophila melanogaster )

    The addition of an src myristoylation sequence to the amino terminus of Dakt1 (myrDakt1) made it constitutively active. UAS drives myrDakt1.

  • y, dAMPKalpha Delta39, FRT19A ( Drosophila melanogaster )

    The dAMPKalpha Delta39 mutation was generated by imprecise excision of the P element P[SUPor-P]KG09204 (Bloomington), and the dAMPKalpha Delta39, FRT19A allele was generated by recombination.

Reagents

  • Akt1 targeting vector ( Construct )

    The Akt1 gene spanning from intron 3 to intron 13 was obtained by screening of a mouse genomic library (lambda FIXII Library; Stratagene). After digestion with SmaI and HindIII, a 1.5-kb fragment of intron 3 was treated with Klenow enzyme and subcloned into the preblunted XbaI site of the loxP-flanked neomycin resistance/thymidine kinase selection cassette vector (pInt3Akt1-loxPneo/TK). A 1.5-kb fragment, which spanned intron 3 to intron 5 and was obtained from Akt1 genomic DNA by digestion with HindIII, was treatment with Klenow enzyme and subcloned into the loxP cassette vector (plox-Akt1ex4-5). A 3-kb fragment obtained from Akt1 genomic DNA by digestion with HindIII and SstI, which spanned intron 5 to intron 12, was treated with Klenow enzyme and inserted into the preblunted ClaI site of plox-Akt1ex4-5 (plox-Akt1ex4-12). After digesting plox-Akt1ex4-12 with SalI and HindIII, a 4.5-kb fragment was inserted between the SalI and HindIII sites of pInt3Akt1-loxPneo/TK.


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Last updated: 2015-09-28T09:42:37.194-04:00

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