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Stowers Institute for Medical Research

8 ARTICLES PUBLISHED IN JoVE

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Neuroscience

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor
Limei Ma 1, Sachiko Haga-Yamanaka 1, Qingfeng Elden Yu 1, Qiang Qiu 1, SangSeong Kim 1, C. Ron Yu 1,2
1Stowers Institute for Medical Research, 2Department of Anatomy and Cell Biology, The University of Kansas School of Medicine

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

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Biology

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation
Otto C. Guedelhoefer IV 1,2, Alejandro Sánchez Alvarado 3,4
1Department of Neurobiology and Anatomy, University of Utah School of Medicine, 2Department of Molecular, Cellular and Developmental Biology, UCSB, 3Howard Hughes Medical Institute, 4Stowers Institute for Medical Research

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

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JoVE Journal

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes
Lu Chen 1,2, Soon-Keat Ooi 1, Ronald C. Conaway 1,2, Joan W. Conaway 1,2
1Stowers Institute for Medical Research, 2Department of Biochemistry & Molecular Biology, Kansas University Medical Center

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

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Biology

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes
Lu Chen 1,2, Soon-Keat Ooi 1, Joan W. Conaway 1,2, Ronald C. Conaway 1,2
1Stowers Institute for Medical Research, 2Department of Biochemistry & Molecular Biology, Kansas University Medical Center

Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

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Biology

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus
Robert Peuß *1, Zachary Zakibe *1, Jaya Krishnan 1, M. Shane Merryman 1, Diana P. Baumann 1, Nicolas Rohner 1,2
1Stowers Institute for Medical Research, 2Department of Molecular & Integrative Physiology, KU Medical Center

In vitro fertilization is a commonly used technique with a variety of model organisms to maintain lab populations and produce synchronized embryos for downstream applications. Here, we present a protocol that implements this technique for different populations of the Mexican tetra fish, Astyanax mexicanus.

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JoVE Journal

Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events
Melvin Noe Gonzalez 1, Joan W. Conaway 1,2, Ronald C. Conaway 1,2
1Stowers Institute for Medical Research, 2Department of Biochemistry and Molecular Biology, Kansas University Medical Center

Here, we describe the assembly of RNA polymerase II (Pol II) elongation complexes requiring only short synthetic DNA and RNA oligonucleotides and purified Pol II. These complexes are useful for studying mechanisms underlying co-transcriptional processing of transcripts associated with the Pol II elongation complex.

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Biology

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry
Shriram Venkatesan *1, Tejbir S. Kandola *1, Alejandro Rodríguez-Gama 1, Andrew Box 1, Randal Halfmann 1,2
1Stowers Institute for Medical Research, 2Department of Molecular and Integrative Physiology, The University of Kansas School of Medicine

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

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Biology

Planarian Ovary Dissection for Ultrastructural Analysis and Antibody Staining
Fengli Guo 1, Melainia McClain 1, Xia Zhao 1, Kexi Yi 1, Tari Parmely 1, Jay Unruh 1, Brian Slaughter 1, Leonid Kruglyak 2,3, Longhua Guo 2,3, Alejandro Sánchez Alvarado 1,2
1Stowers Institute for Medical Research, 2Howard Hughes Medical Institute, 3Department of Human Genetics, University of California Los Angeles

This protocol presents steps taken to dissect ovaries in the freshwater planarians, Schmidtea mediterranea. The dissected ovaries are compatible for antibody immunostaining and ultrastructural analysis with transmission electron microscopy to study the cell biology of the oocytes and somatic cells, providing an imaging depth and quality that were previously inaccessible.

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