1Molecular, Cellular and Developmental Biology Graduate Program, The Ohio State University, 2Center for Cardiovascular and Pulmonary Research, The Heart Center, The Research Institute at Nationwide Children's Hospital, 3Department of Pediatrics, The Ohio State University
The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.
Published August 21, 2014. Keywords: Cellular Biology, Heart valve, Valve Endothelial Cells (VEC), Fluorescence Activated Cell Sorting (FACS), Mouse, Embryo, Adult, GFP.
1Département de Génomique Fonctionnelle et Cancer, Institut de Génétique et de Biologie Moléculaire et Cellulaire, 2Center for Life NanoScience, Istituto Italiano di Tecnologia
The aim of this publication is to visualize and discuss the operative steps of an Enhanced Northern Blot protocol on RNA extracted from Drosophila melanogaster embryos, cells, and tissues. This protocol is particularly useful for the efficient detection of small RNA species.
Published August 21, 2014. Keywords: Molecular Biology, Northern blotting, Noncoding RNAs, microRNAs, rasiRNA, Gene expression, Gcm/Glide, Drosophila melanogaster
1Stem Cell Research Center, Department of Bioengineering and Orthopedic Surgery, University of Pittsburgh, 2Department of Orthopedic Surgery, University of Pittsburgh, 3Nazarbayev University Research and Innovation System, Nazarbayev University, 4Department of Orthopaedic Surgery, UCLA Orthopaedic Hospital and the Orthopaedic Hospital Research Center, University of California at Los Angeles, 5Department of Cell Biology, Erasmus MC Stem Cell Institute, 6OHSU Center for Regenerative Medicine, Oregon Health & Science University, 7Centre for Cardiovascular Science and MRC Centre for Regenerative Medicine, Queen's Medical Research Institute and University of Edinburgh, 8David Geffen School of Medicine and the Orthopaedic Hospital Research Center, University of California at Los Angeles, 9Stem Cell Research Center, Department of Orthopedic Surgery and McGowan Institute for Regenerative Medicine, University of Pittsburgh
Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.
Published August 21, 2014. Keywords: Cellular Biology, Blood Vessel; Pericyte; Adventitial Cell; Myogenic Endothelial Cell; Multipotent Precursor
The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.
Published August 17, 2014. Keywords: Genetics, gene regulation, transcriptional regulation, sequence-activity mapping, reporter assay, library cloning, transfection, tag sequencing, mammalian cells
1Department of Biology, College of William & Mary
Immunostaining is an effective technique for visualizing specific cell types and proteins within tissues. By utilizing sonication, the protocol described here alleviates the need to dissect Drosophila melanogaster tissues from late-stage embryos and larvae before immunostaining. We provide an efficient methodology for the immunostaining of formaldehyde-fixed whole mount larvae.
Published August 14, 2014. Keywords: Molecular Biology, Drosophila, embryo, larvae, sonication, fixation, immunostain, immunofluorescence, organogenesis, development
1The Ritchie Centre, Monash Institute of Medical Research, 2Department of Obstetrics and Gynaecology, Monash Medical Centre, 3Animal Resource Centre, Perth, Australia, 4Wake Forest Institute for Regenerative Medicine
The assessment of respiratory physiology has traditionally relied upon techniques, which require restraint or sedation of the animal. Unrestrained whole-body plethysmography, however, provides precise, non-invasive, quantitative analysis of respiratory physiology in animal models. In addition, the technique allows repeated respiratory assessment of mice allowing for longitudinal studies.
Published August 12, 2014. Keywords: Physiology, Unrestrained Whole Body Plethysmography, Lung function, Respiratory Disease, Rodents
1Department of Genetics, University of Cambridge, 2Wellcome Trust - Medical Research Council Stem Cell Institute, University of Cambridge
This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.
Published August 11, 2014. Keywords: Genetics, Retrovirus, Lentivirus, Organoid culture, Lgr5, Intestine, 3Rs
1Department of Biological Sciences, University of Notre Dame
The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.
Published August 9, 2014. Keywords: Cellular Biology, zebrafish; kidney; nephron; nephrology; renal; regeneration; proximal tubule; distal tubule; segment; mesonephros; physiology; acute kidney injury (AKI)
1Light Microscopy Core Facility, NHLBI/NIH, 2Hematology Branch, NHLBI/NIH
Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.
Published August 6, 2014. Keywords: Stem Cell Biology, LeGO imaging, clonal tracking, fluorescent proteins, confocal microscopy, multiphoton microscopy, hematopoiesis, lentiviral vectors, hematopoietic stem cells
1Environmental Health Science and Research Bureau, Health Canada, Environmental Health Centre
De novo mutations in the male germline may contribute to adverse health outcomes in subsequent generations. Here we describe a protocol for the use of a transgenic rodent model for quantifying mutations in male germ cells induced by environmental agents.
Published August 6, 2014. Keywords: Genetics, sperm, spermatogonia, male germ cells, spermatogenesis, de novo mutation, OECD TG 488, transgenic rodent mutation assay, N-ethyl-N-nitrosourea, genetic toxicology