The Experts below are selected from a list of 321 Experts worldwide ranked by ideXlab platform

Edward J. Kelly - One of the best experts on this subject based on the ideXlab platform.

  • Development and characterization of transgenic mouse models for Conditional Gene Knockout in the blood–brain and blood-CSF barriers
    Transgenic Research, 2012
    Co-Authors: Matthew H. Crouthamel, Edward J. Kelly
    Abstract:

    For many CNS acting drugs, penetration into the central nervous system (CNS) is limited by the blood-CNS-barriers. In an effort to quantitate the role of the protein components that make up the blood-CNS-barriers, we created transgenic mice that allow Conditional Gene Knockout using Cre/ loxP technology. We targeted the expression of Cre-recombinase to the choroid plexus (the blood-cerebral spinal fluid barrier) using the lymphotropic papovavirus control region ( LPVcr ) and to brain endothelium (the blood–brain-barrier) using the proximal promoter region of the human von Willebrand Factor Gene ( hVWF - f ). We verified that LPVcr restricts expression to the choroid plexus in adult mice by using the LPVcr to drive n - LacZ expression in transgenic mice. The LPV-Cre and hVWF-Cre plasmids were then constructed and tested for Cre-recombinase function in vitro, and subsequently used to create transgenic mice. The resulting transgenic mice were characterized for cell-type specific Cre-mediated endonuclease activity by crossing them with transgenic mice containing a loxP-flanked-LacZ/EGFP dual reporter Gene Z/EG. The dual Cre-Z/EG transgenic offspring were evaluated for the location of EGFP mRNA expression by reverse transcriptase PCR and for protein expression by immunohistochemistry. Immunohistochemistry for EGFP verified expression in the target cells, and no ectopic expression outside of the expected cell types. The LPV-Cre.0607 transgenic line expressed functional Cre only in the choroid plexus and hVWF-Cre.1304 line in brain endothelium.

  • development and characterization of transgenic mouse models for Conditional Gene Knockout in the blood brain and blood csf barriers
    Transgenic Research, 2012
    Co-Authors: Matthew H. Crouthamel, Edward J. Kelly
    Abstract:

    For many CNS acting drugs, penetration into the central nervous system (CNS) is limited by the blood-CNS-barriers. In an effort to quantitate the role of the protein components that make up the blood-CNS-barriers, we created transgenic mice that allow Conditional Gene Knockout using Cre/loxP technology. We targeted the expression of Cre-recombinase to the choroid plexus (the blood-cerebral spinal fluid barrier) using the lymphotropic papovavirus control region (LPVcr) and to brain endothelium (the blood–brain-barrier) using the proximal promoter region of the human von Willebrand Factor Gene (hVWF-f). We verified that LPVcr restricts expression to the choroid plexus in adult mice by using the LPVcr to drive n-LacZ expression in transgenic mice. The LPV-Cre and hVWF-Cre plasmids were then constructed and tested for Cre-recombinase function in vitro, and subsequently used to create transgenic mice. The resulting transgenic mice were characterized for cell-type specific Cre-mediated endonuclease activity by crossing them with transgenic mice containing a loxP-flanked-LacZ/EGFP dual reporter Gene Z/EG. The dual Cre-Z/EG transgenic offspring were evaluated for the location of EGFP mRNA expression by reverse transcriptase PCR and for protein expression by immunohistochemistry. Immunohistochemistry for EGFP verified expression in the target cells, and no ectopic expression outside of the expected cell types. The LPV-Cre.0607 transgenic line expressed functional Cre only in the choroid plexus and hVWF-Cre.1304 line in brain endothelium.

Christian Flueck - One of the best experts on this subject based on the ideXlab platform.

  • The Plasmodium falciparum Artemisinin Susceptibility-Associated AP-2 Adaptin μ Subunit is Clathrin Independent and Essential for Schizont Maturation
    mBio, 2020
    Co-Authors: Ryan C. Henrici, Rachel L. Edwards, Martin Zoltner, Donelly A. Van Schalkwyk, Melissa N. Hart, Franziska Mohring, Robert W. Moon, Stephanie D. Nofal, Avnish Patel, Christian Flueck
    Abstract:

    The efficacy of current antimalarial drugs is threatened by reduced susceptibility of Plasmodium falciparum to artemisinin, associated with mutations in pfkelch13 Another Gene with variants known to modulate the response to artemisinin encodes the μ subunit of the AP-2 adaptin trafficking complex. To elucidate the cellular role of AP-2μ in P. falciparum, we performed a Conditional Gene Knockout, which severely disrupted schizont organization and maturation, leading to mislocalization of key merozoite proteins. AP-2μ is thus essential for blood-stage replication. We Generated transgenic P. falciparum parasites expressing hemagglutinin-tagged AP-2μ and examined cellular localization by fluorescence and electron microscopy. Together with mass spectrometry analysis of coimmunoprecipitating proteins, these studies identified AP-2μ-interacting partners, including other AP-2 subunits, the K10 kelch-domain protein, and PfEHD, an effector of endocytosis and lipid mobilization, but no evidence was found of interaction with clathrin, the expected coat protein for AP-2 vesicles. In reverse immunoprecipitation experiments with a clathrin nanobody, other heterotetrameric AP-complexes were shown to interact with clathrin, but AP-2 complex subunits were absent.IMPORTANCE We examine in detail the AP-2 adaptin complex from the malaria parasite Plasmodium falciparum In most studied organisms, AP-2 is involved in bringing material into the cell from outside, a process called endocytosis. Previous work shows that changes to the μ subunit of AP-2 can contribute to drug resistance. Our experiments show that AP-2 is essential for parasite development in blood but does not have any role in clathrin-mediated endocytosis. This suggests that a specialized function for AP-2 has developed in malaria parasites, and this may be important for understanding its impact on drug resistance.

Brian Sauer - One of the best experts on this subject based on the ideXlab platform.

  • Conditional Gene Knockout using cre recombinase.
    Methods in molecular biology (Clifton N.J.), 2000
    Co-Authors: Brian Sauer
    Abstract:

    Cre recombinase has become an important instrument for achieving precise Genetic manipulation in mice. Many of these desired Genetic manipulations rely on Cre’s ability to direct spatially and temporally specified excision of a predesignated DNA sequence that has been flanked by directly repeated copies of the loxP recombination site. Success in achieving such Conditional mutaGenesis in mice depends both on the careful design of Conditional alleles and on reliable detection of cre Gene expression. These procedures include PCR, immunohistochemistry and the use of a recombination-proficient GFP-tagged Cre protein.

  • Brief expression of a GFPcre fusion Gene in embryonic stem cells allows rapid retrieval of site-specific genomic deletions
    Nucleic acids research, 1997
    Co-Authors: Sara Gagneten, Jeffrey S. Miller, Brian Sauer
    Abstract:

    The Cre DNA recombinase of bacteriophage P1 has become a useful tool for precise genomic manipulation in embryonic stem (ES) cells that have been Gene modified by homologous recombination. We have re-engineered the cre Gene to allow ready identification of living Cre+cells by constructing a functional fusion between Cre and an enhanced green fluorescent protein from Aequorea victoria (GFPS65T). The GFP cre fusion Gene product rapidly targeted the nucleus in the absence of any exogenous nuclear localization signal. Moreover, GFPCre catalyzed efficient DNA recombination in both a mouse 3T3 derivative cell line and in murine ES cells. Fluorescence- activated cell sorting (FACS) of transiently GFP cre -transfected ES cells not only allowed rapid and efficient isolation of Cre+cells after DNA transfection but also demonstrated that a burst of Cre expression is sufficient to commit cells to Cre-mediated 'pop-out' of loxP -tagged DNA from the genome. Thus, GFP cre allows rapid identification of living cells in which loxP - flanked DNA sequences are destined to be removed from the genome by Cre-mediated recombination without reliance on recombinational activation or inactivation of a marker Gene at the target locus. In addition, the GFP cre fusion Gene will prove useful in tracing tissue-specific Cre expression in transgenic animals, thereby facilitating the Generation and analysis of Conditional Gene Knockout mice.

Matthew H. Crouthamel - One of the best experts on this subject based on the ideXlab platform.

  • Development and characterization of transgenic mouse models for Conditional Gene Knockout in the blood–brain and blood-CSF barriers
    Transgenic Research, 2012
    Co-Authors: Matthew H. Crouthamel, Edward J. Kelly
    Abstract:

    For many CNS acting drugs, penetration into the central nervous system (CNS) is limited by the blood-CNS-barriers. In an effort to quantitate the role of the protein components that make up the blood-CNS-barriers, we created transgenic mice that allow Conditional Gene Knockout using Cre/ loxP technology. We targeted the expression of Cre-recombinase to the choroid plexus (the blood-cerebral spinal fluid barrier) using the lymphotropic papovavirus control region ( LPVcr ) and to brain endothelium (the blood–brain-barrier) using the proximal promoter region of the human von Willebrand Factor Gene ( hVWF - f ). We verified that LPVcr restricts expression to the choroid plexus in adult mice by using the LPVcr to drive n - LacZ expression in transgenic mice. The LPV-Cre and hVWF-Cre plasmids were then constructed and tested for Cre-recombinase function in vitro, and subsequently used to create transgenic mice. The resulting transgenic mice were characterized for cell-type specific Cre-mediated endonuclease activity by crossing them with transgenic mice containing a loxP-flanked-LacZ/EGFP dual reporter Gene Z/EG. The dual Cre-Z/EG transgenic offspring were evaluated for the location of EGFP mRNA expression by reverse transcriptase PCR and for protein expression by immunohistochemistry. Immunohistochemistry for EGFP verified expression in the target cells, and no ectopic expression outside of the expected cell types. The LPV-Cre.0607 transgenic line expressed functional Cre only in the choroid plexus and hVWF-Cre.1304 line in brain endothelium.

  • development and characterization of transgenic mouse models for Conditional Gene Knockout in the blood brain and blood csf barriers
    Transgenic Research, 2012
    Co-Authors: Matthew H. Crouthamel, Edward J. Kelly
    Abstract:

    For many CNS acting drugs, penetration into the central nervous system (CNS) is limited by the blood-CNS-barriers. In an effort to quantitate the role of the protein components that make up the blood-CNS-barriers, we created transgenic mice that allow Conditional Gene Knockout using Cre/loxP technology. We targeted the expression of Cre-recombinase to the choroid plexus (the blood-cerebral spinal fluid barrier) using the lymphotropic papovavirus control region (LPVcr) and to brain endothelium (the blood–brain-barrier) using the proximal promoter region of the human von Willebrand Factor Gene (hVWF-f). We verified that LPVcr restricts expression to the choroid plexus in adult mice by using the LPVcr to drive n-LacZ expression in transgenic mice. The LPV-Cre and hVWF-Cre plasmids were then constructed and tested for Cre-recombinase function in vitro, and subsequently used to create transgenic mice. The resulting transgenic mice were characterized for cell-type specific Cre-mediated endonuclease activity by crossing them with transgenic mice containing a loxP-flanked-LacZ/EGFP dual reporter Gene Z/EG. The dual Cre-Z/EG transgenic offspring were evaluated for the location of EGFP mRNA expression by reverse transcriptase PCR and for protein expression by immunohistochemistry. Immunohistochemistry for EGFP verified expression in the target cells, and no ectopic expression outside of the expected cell types. The LPV-Cre.0607 transgenic line expressed functional Cre only in the choroid plexus and hVWF-Cre.1304 line in brain endothelium.

Joe Warren - One of the best experts on this subject based on the ideXlab platform.