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Sanford Burnham Prebys Medical Discovery Institute

11 ARTICLES PUBLISHED IN JoVE

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Biology

Fluorescent Labeling of Drosophila Heart Structures
Nakissa N. Alayari 1,2, Georg Vogler 2, Ouarda Taghli-Lamallem 2, Karen Ocorr 2, Rolf Bodmer 2, Anthony Cammarato 1,2
1Biology Department, San Diego State University, 2Development and Aging Program, NASCR Center, The Sanford Burnham Institute for Medical Research

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

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Biology

Semi-automated Optical Heartbeat Analysis of Small Hearts
Karen Ocorr 1, Martin Fink 2, Anthony Cammarato 1,3, Sanford I. Bernstein 3, Rolf Bodmer 1
1Development and Aging Program, The Sanford Burnham Institute for Medical Research, 2Cardiac Electrophysiology Group, Dept. of Physiology, Anatomy and Genetics, The Sanford Burnham Institute for Medical Research, 3Biology Department and Heart Institute, San Diego State University

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

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Neuroscience

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction
Xuejun H. Parsons 1,2, Yang D. Teng 3,4, James F. Parsons 1,2, Evan Y. Snyder 1,2,5, David B. Smotrich 1,2,6, Dennis A. Moore 1,2
1San Diego Regenerative Medicine Institute, 2Xcelthera, 3Department of Neurosurgery, Harvard Medical School, 4Division of SCI Research, VA Boston Healthcare System, 5Program in Stem Cell & Regenerative Biology, Sanford-Burnham Medical Research Institute, 6La Jolla IVF

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

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Biology

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction
Xuejun H. Parsons 1,2, Yang D. Teng 3,4, James F. Parsons 1,2, Evan Y. Snyder 1,2,5, David B. Smotrich 1,2,6, Dennis A. Moore 1,2
1San Diego Regenerative Medicine Institute, 2Xcelthera, 3Department of Neurosurgery, Harvard Medical School, 4Division of SCI Research, VA Boston Healthcare System, 5Program in Stem Cell & Regenerative Biology, Sanford-Burnham Medical Research Institute, 6La Jolla IVF

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

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Genetics

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster
Soda Balla Diop 1, Ryan T. Birse 1, Rolf Bodmer 1
1Development, Aging and Regeneration Program, Sanford Burnham Prebys Medical Discovery Institute

This is a high fat diet feeding protocol to induce obesity in Drosophila, a model for understanding fundamental molecular mechanisms implicated in lipotoxicity. It also provides a high throughput triacylglyceride assay for measuring fat accumulation in Drosophila and potentially other (insect) models under various dietary, environmental, genetic or physiological conditions.

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Developmental Biology

Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells
Michael S. Yu *1,2, Sean Spiering *1, Alexandre R. Colas 1
1Sanford Burnham Prebys Medical Discovery Institute, 2Department of Bioengineering, University of California at San Diego

Here we describe a scalable method, using a simple combination of Activin A and lentivirus-mediated Id1-overexpression, to generate first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells.

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Cancer Research

Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays
Anagha Deshpande 1, Bo Rui Chen 1, Luyi Zhao 1, Kelsey Saddoris 1, Mayuri Kerr 1, Nan Zhu 2, Prashant Mali 3, Aniruddha J. Deshpande 1
1Tumor Initiation and Maintenance Program, Sanford Burnham Prebys Medical Discovery Institute, 2Blood Research Institute, Blood Center of Wisconsin, 3Department of Bioengineering, University of California, San Diego

This manuscript describes a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR-Cas9-based method for simple and expeditious investigation of the role of multiple candidate genes in Acute Myeloid Leukemia (AML) cell proliferation in parallel. This technique is scalable and can be applied in other cancer cell lines as well.

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Genetics

Tumorsphere Derivation and Treatment from Primary Tumor Cells Isolated from Mouse Rhabdomyosarcomas
Francesca Boscolo Sesillo 1,2,3, Alessandra Sacco 2
1Graduate School of Biomedical Sciences, Sanford Burnham Prebys Medical Discovery Institute, 2Development, Aging and Regeneration Program, Sanford Burnham Prebys Medical Discovery Institute, 3Department of Obstetrics, Gynecology, and Reproductive Sciences, Division of Female Pelvic Medicine and Reconstructive Surgery, University California San Diego

This protocol describes a reproducible method for isolation of mouse rhabdomyosarcoma primary cells, tumorsphere formation and treatment, and allograft transplantation starting from tumorspheres cultures.

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Cancer Research

Assessing Cellular Target Engagement by SHP2 (PTPN11) Phosphatase Inhibitors
Lester J. Lambert 1, Celeste Romero 1, Douglas J. Sheffler 1, Maria Celeridad 1, Nicholas D. P. Cosford 1, Lutz Tautz 1
1Cancer Metabolism & Signaling Networks Program, NCI-Designated Cancer Center, Sanford Burnham Prebys Medical Discovery Institute

The ability to assess target engagement by candidate inhibitors in intact cells is crucial for drug discovery. This protocol describes a 384 well format cellular thermal shift assay that reliably detects cellular target engagement of inhibitors targeting either wild-type SHP2 or its oncogenic variants.

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Developmental Biology

Generation of 3D Whole Lung Organoids from Induced Pluripotent Stem Cells for Modeling Lung Developmental Biology and Disease
Sandra L. Leibel 1,2,3, Rachael N. McVicar 2,3, Alicia M. Winquist 2,3, Evan Y. Snyder 1,2,3
1Department of Pediatrics, University of California, San Diego School of Medicine, 2Sanford Consortium for Regenerative Medicine, 3Sanford Burnham Prebys Medical Discovery Institute

The article describes step wise directed differentiation of induced pluripotent stem cells to three-dimensional whole lung organoids containing both proximal and distal epithelial lung cells along with mesenchyme.

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Neuroscience

Quantification of Immunostained Caspase-9 in Retinal Tissue
Crystal K. Colón Ortiz 1, Anna M. Potenski 2, Kendra V. Johnson 1, Claire W. Chen 1, Scott J. Snipas 3, Ying Y. Jean 1, Maria I. Avrutsky *1, Carol M. Troy *1,4,5
1Department of Pathology & Cell Biology, Vagelos College of Physicians and Surgeons, Columbia University, 2Department of Molecular Pharmacology and Therapeutics, Vagelos College of Physicians and Surgeons, Columbia University, 3NCI-designated Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, 4Department of Neurology, Vagelos College of Physicians and Surgeons, Columbia University, 5The Taub Institute for Research on Alzheimer’s Disease and the Aging Brain, Vagelos College of Physicians and Surgeons, Columbia University

Presented here is a detailed immunohistochemistry protocol to identify, validate, and target functionally relevant caspases in complex tissues.

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