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
1Department of Bioengineering, University of Pennsylvania, 2Integrated DNA Technologies, Inc.
Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.
Published August 6, 2014. Keywords: Genetics, RNA, imaging, single molecule, fluorescence, living cell
1Graduate School of Medicine, Jichi Medical University, 2Division of Biology, Center for Molecular Medicine, Jichi Medical University, 3Department of Developmental Neuroscience, United Center for Advanced Research and Translational Medicine (ART), Tohoku University School of Medicine
Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.
Published August 3, 2014. Keywords: Physiology, WEC, male rats, abdominal aorta, rat serum, fibrin clot, hemolysis
1Department of Animal and Avian Sciences, University of Maryland, 2Department of Cell Biology and Molecular Genetics, University of Maryland
C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.
Published August 2, 2014. Keywords: Molecular Biology, C. elegans, axenic media, transgenics, microparticle bombardment, heme, nutrition