University of Minnesota View Institution's Website 80 articles published in JoVE Biochemistry Sample Preparation for In Situ Cryotomography of Mammalian Cells Noah Weber*1,2, Brennan Hinks*2,3, Jacob Jensen2, Thomas Lidahl2,3, Luiza Mendonça2 1College of Liberal Arts, University of Minnesota, 2Department of Biochemistry, Molecular Biology and Biophysics, Medical School, University of Minnesota, 3College of Biological Sciences, University of Minnesota This method provides an accessible and flexible protocol for the preparation of electron microscopy (EM) grids for in situ cellular cryotomography and correlative light and electron microscopy (CLEM). Medicine Surgical Porcine Model of Chronic Myocardial Ischemia Treated by Exosome-laden Collagen Patch and Off-pump Coronary Artery Bypass Graft Rishav Aggarwal1, Annie Shao1, Koray N. Potel2, Laura Hocum Stone1, Cory Swingen1, Christin Wright1, Edward O. McFalls3, Tammy A. Butterick4,5, Rosemary F. Kelly1 1Department of Surgery, University of Minnesota, 2 This study presents a surgical porcine model of chronic myocardial ischemia due to progressive coronary artery stenosis, resulting in impaired cardiac function without infarction. Following ischemia, animals undergo off-pump coronary artery bypass graft with epicardial placement of stem cells-derived exosomes-laden collagen patch. This adjunctive therapy improves myocardial function and recovery. Medicine Biobanking of Human Aqueous and Vitreous Liquid Biopsies for Molecular Analyses Julian Wolf1,2, Teja Chemudupati1,2, Aarushi Kumar1,2, Ditte K. Rasmussen1,2, Karen M. Wai2, Robert T. Chang2, Artis A. Montague2, Peter H. Tang3,4, Alexander G. Bassuk5,6,7, Antoine Dufour8,9, Prithvi Mruthrunjaya2, Vinit B. Mahajan1,2,10 1Molecular Surgery Laboratory, Stanford University, 2Department of Ophthalmology, Byers Eye Institute, Stanford University, 3Department of Ophthalmology and Visual Neurosciences, University of Minnesota, 4Retina Consultants of Minnesota, 5Department of Pediatrics, University of Iowa, 6Department of Neurology, University of Iowa, 7The Iowa Neuroscience Institute (INI), University of Iowa, 8Department of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, 9Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, 10Veterans Affairs Palo Alto Health Care System This protocol presents an integrated biorepository platform for the standardized collection, annotation, and biobanking of high-quality human aqueous humor and vitreous liquid biopsies for molecular downstream analyses, including proteomics, metabolomics, and glycomics. Biology Rearing the Cabbage White Butterfly (Pieris rapae) in Controlled Conditions: A Case Study with Heavy Metal Tolerance Emilie C. Snell-Rood1, Megan E. Kobiela1,2 1Deptartment of Ecology, Evolution and Behavior, University of Minnesota, 2Biology, Sweet Briar College This paper presents a detailed protocol for rearing the cabbage white butterfly in controlled lab conditions with an artificial diet, which allows precise manipulations of early-life nutrition and toxin exposure. The representative results show how heavy metal toxicity can be assayed with this protocol. Biology Evidence for EpCAM and Cytokeratin Expressing Epithelial Cells in Normal Human and Murine Blood and Bone Marrow Stephanie M. Holtorf1, Jennifer Boyle1, Rebecca Morris1 1The Hormel Institute, University of Minnesota This paper presents a reproducible method with new findings on the presence of epithelial cells in normal human and mouse blood and bone marrow using flow cytometry and immunofluorescence microscopy. Krt1-14;mTmG transgenic mice were used as an in vivo method to confirm these findings. Biology Tick Artificial Membrane Feeding for Ixodes scapularis Benedict Khoo1, Benjamin Cull2, Jonathan D. Oliver1 1Division of Environmental Health Sciences, School of Public Health, University of Minnesota, 2Department of Entomology, College of Food, Agricultural and Natural Resources, University of Minnesota Presented here is a method to blood feed ticks in vitro via an artificial membrane system to allow for partial or full engorgement of a variety of tick life stages. Biology An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines Xin-Ru Wang1, Nicole Y. Burkhardt1, Lisa D. Price1, Ulrike G. Munderloh1 1Department of Entomology, University of Minnesota Electroporation is a rapid, broadly adopted method for introducing exogenous DNA into the genus Rickettsia. This protocol provides a useful electroporation method for the transformation of obligate intracellular bacteria in the genus Rickettsia. Neuroscience Imaging the Aging Cochlea with Light-Sheet Fluorescence Microscopy Peter A. Santi1, Shane B. Johnson1 1Department of Otolaryngology, University of Minnesota A light-sheet microscope was developed to image and digitize whole cochlea. Engineering Microtensiometer for Confocal Microscopy Visualization of Dynamic Interfaces Steven V. Iasella1, Sourav Barman1, Clara Ciutara1, Boxun Huang1, Michael L. Davidson2, Joseph A. Zasadzinski1 1Department of Chemical Engineering and Materials Science, University of Minnesota, 2Department of Chemical Engineering, Carnegie Mellon University This manuscript describes the design and operation of a microtensiometer/confocal microscope to do simultaneous measurements of interfacial tension and surface dilatational rheology while visualizing the interfacial morphology. This provides the real-time construction of structure-property relationships of interfaces important in technology and physiology. Developmental Biology Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures Bridget T. Jacques-Fricke1, Julaine Roffers-Agarwal2,3, Callie M. Gustafson2,3, Laura S. Gammill2,3 1Department of Biology and Neuroscience Program, Hamline University, 2Department of Genetics, Cell Biology and Development, University of Minnesota, 3Developmental Biology Center, University of Minnesota This versatile protocol describes the isolation of premigratory neural crest cells (NCCs) through the excision of cranial neural folds from chick embryos. Upon plating and incubation, migratory NCCs emerge from neural fold explants, allowing for assessment of cell morphology and migration in a simplified 2D environment. Biology In Vivo Measurement of Hindlimb Dorsiflexor Isometric Torque from Pig Benjamin T. Corona1, Jarrod A. Call2,3, Matthew Borkowski4, Sarah M. Greising5 1School of Medicine, Wake Forest University, 2Department of Kinesiology, University of Georgia, 3Regenerative Bioscience Center, University of Georgia, 4Aurora Scientific Inc., 5School of Kinesiology, University of Minnesota The present protocol describes concise experimental details on the evaluation and interpretation of in vivo torque data obtained via electrical stimulation of the common peroneal nerve in anesthetized pigs. Bioengineering Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection Gabriella Shull*1,2, Christiane Haffner*3, Wieland B. Huttner3, Elena Taverna3,4, Suhasa B. Kodandaramaiah1,5,6 1Department of Biomedical Engineering, University of Minnesota, 2Department of Biomedical Engineering, Duke University, 3Max Planck Institute of Molecular Cell Biology and Genetics, 4Max Planck Institute for Evolutionary Anthropology, 5Department of Mechanical Engineering, University of Minnesota, 6Graduate Program in Neuroscience, University of Minnesota This protocol demonstrates the use of a robotic platform for microinjection into single neural stem cells and neurons in brain slices. This technique is versatile and offers a method of tracking cells in tissue with high spatial resolution. Neuroscience Cerebellar Regional Dissection for Molecular Analysis Katherine A. Hamel1, Marija Cvetanovic1 1Department of Neuroscience, University of Minnesota Different cerebellar regions have been implicated to play a role in distinct behavioral outputs, yet the underlying molecular mechanisms remain unknown. This work describes a method to reproducibly and quickly dissect cerebellar cortex of the hemispheres, anterior and posterior regions of the vermis, and the deep cerebellar nuclei in order to probe for molecular differences by isolating RNA and testing for differences in gene expression. Biology Genome Engineering of Primary Human B Cells Using CRISPR/Cas9 Kanut Laoharawee1,2,3, Matthew J. Johnson1,2,3, Walker S. Lahr1,2,3, Joseph J. Peterson1,2,3, Beau R. Webber1,2,3, Branden S. Moriarity1,2,3 1Department of Pediatrics, University of Minnesota, 2Center for Genomic Engineering, University of Minnesota, 3Masonic Cancer Center, University of Minnesota Here we provide a detailed, step-by-step protocol for CRISPR/Cas9-based genome engineering of primary human B cells for gene knockout (KO) and knock-in (KI) to study biological functions of genes in B cells and the development of B-cell therapeutics. Biology Isolation of Cardiomyocytes from Fixed Hearts for Immunocytochemistry and Ploidy Analysis Doğacan Yücel*1,2, Jacob Solinsky*2, Jop H. van Berlo1,2,3 1Department of Integrative Biology and Physiology, University of Minnesota, 2Lillehei Heart Institute, Department of Medicine, University of Minnesota, 3Stem Cell Institute, University of Minnesota The goal of this work is to develop a method to reproducibly isolate cardiomyocytes from the adult heart and measure DNA content and nucleation. Environment Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis Jared A. Spackman1, Fabian G. Fernandez1 1Department of Soil, Water and Climate, University of Minnesota A microplot design for 15N tracer research is described to accommodate multiple in-season plant and soil sampling events. Soil and plant sample collection and processing procedures, including grinding and weighing protocols, for 15N analysis are put forth. Bioengineering Transduction and Expansion of Primary T Cells in Nine Days with Maintenance of Central Memory Phenotype Mary S. Pampusch1, Pamela J. Skinner1 1Department of Veterinary and Biomedical Sciences, University of Minnesota We outline a 9-day protocol for the transduction and expansion of rhesus macaque peripheral blood mononuclear cells which yields cells with excellent co-expression of the genes of interest in sufficient number for infusion studies of cell efficacy. Biochemistry In Vivo Calcium Imaging in C. elegans Body Wall Muscles Ashley A. Martin1,2, Simon Alford3, Janet E. Richmond1 1Department of Biological Sciences, University of Illinois at Chicago, 2Department of Integrative Biology and Physiology, University of Minnesota, 3Department of Anatomy and Cell Biology, University of Illinois at Chicago This method provides a way to couple optogenetics and genetically encoded calcium sensors to image baseline cytosolic calcium levels and changes in evoked calcium transients in the body wall muscles of the model organism C. elegans. Biology Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture Rebecca J. Morris1, Nyssa Readio1, Kelsey Boland1, Kelly Johnson1, Sonali Lad1, Anupama Singh1, Ashok Singh1, Stephanie Holtorf1, Samantha Skaar1 1Hormel Institute, University of Minnesota The goal of this protocol is to isolate epidermal keratinocytes from the dorsal skin of adult mice for a variety of downstream applications such as molecular biology, biochemistry, fluorescence activated cell sorting, and primary in vitro uses (e.g., clonogenic keratinocytes). Biology SA-β-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs Heike Fuhrmann-Stroissnigg*1, Fernando E. Santiago*1,2,3, Diego Grassi1, YuanYuan Ling1, Laura J. Niedernhofer1,2,3, Paul D. Robbins1,2,3 1Department of Molecular Medicine and the Center on Aging, The Scripps Research Institute, 2Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 3Institute on the Biology of Aging and Metabolism, University of Minnesota Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated β-Galactosidase activity in single cells. Medicine Spontaneous and Evoked Measures of Pain in Murine Models of Monoarticular Knee Pain Hollis E. Krug1,2, Christopher Dorman2, Nicole Blanshan2, Sandra Frizelle2, Maren Mahowald1,2 1Department of Medicine, University of Minnesota, 2Research Department, VA Health Care Center We have developed an evoked measure of arthritis pain and coupled it with a standardized method for measuring spontaneous pain in different murine models of chemically induced arthritis. These measures are sensitive and reproducible for different types of joint pain. Engineering Fabrication of Three-Dimensional Graphene-Based Polyhedrons via Origami-Like Self-Folding Daeha Joung1, Daniel Wratkowski1, Chunhui Dai1, Seokhyeong Lee1, Jeong-Hyun Cho1 1Department of Electrical and Computer Engineering, University of Minnesota, Minneapolis, United States Here, we present a protocol for fabrication of 3D graphene-based polyhedrons via origami-like self-folding. Environment Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn (Zea mays) As an Exemplar Carrie A. Deans1,2, Gregory A. Sword1, Paul A. Lenhart3, Eric Burkness2, William D. Hutchison2, Spencer T. Behmer1 1Department of Entomology, Texas A&M University, 2Department of Entomology, University of Minnesota, 3Department of Entomology, University of Kentucky The protocols described herein provide a clear and approachable methodology for measuring soluble protein and digestible (non-structural) carbohydrate content in plant tissues. The ability to quantify these two plant macronutrients has significant implications for advancing the fields of plant physiology, nutritional ecology, plant-herbivore interactions and food-web ecology. Biochemistry Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues Dana M. Freund1, Katherine A. Sammons2, Nokwanda P. Makunga3, Jerry D. Cohen1, Adrian D. Hegeman2 1Department of Horticultural Science, Microbial and Plant Genomics Institute, University of Minnesota, 2Department of Horticultural Science, Department of Plant and Microbial Biology, Microbial and Plant Genomics Institute, University of Minnesota, 3Department of Botany and Zoology, Stellenbosch University Leaf spray mass spectrometry is a direct chemical analysis technique that minimizes the sample preparation and eliminates chromatography, allowing for the rapid detection of small molecules from plant tissues. Medicine Surgical Swine Model of Chronic Cardiac Ischemia Treated by Off-Pump Coronary Artery Bypass Graft Surgery Laura Hocum Stone1, Christin Wright1, Erin Chappuis1, Mia Messer1, Herbert B. Ward1, Edward O. McFalls2, Rosemary F. Kelly1 1Department of Surgery, University of Minnesota, 2Cardiology, Minneapolis VA Medical Center This protocol presents a surgical large animal model of chronic, single vessel ischemia that results in regional abnormalities but does not create infarct, known as hibernating myocardium. Following establishment of chronic ischemia, animals are treated with off-pump LIMA-LAD coronary artery bypass graft surgery to revascularize the ischemic tissue. Genetics A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells Lisa N. Chesner1, Colin Campbell1 1Department of Pharmacology, University of Minnesota The goal of this protocol is to quantify the repair of defined DNA-protein crosslinks on plasmid DNA. Lesioned plasmids are transfected into recipient mammalian cell lines and low-molecular weight harvested at multiple time points post-transfection. DNA repair kinetics are quantified using strand-specific primer extension followed by qPCR. Medicine An Anatomical Study of Nerves at Risk During Minimally Invasive Hallux Valgus Surgery Miki Dalmau-Pastor1,2,3, Jordi Vega1,4, Francesc Malagelada1,5, Fernando Peña6, Maria Cristina Manzanares-Céspedes1 1Laboratory of Arthroscopic and Surgical Anatomy. Department of Pathology and Experimental Therapeutics (Human Anatomy and Embryology Unit), University of Barcelona, 2Health Sciences Faculty of Manresa, University of Vic-Central University of Catalunya, 3Groupe de Recherche et d'Etude en Chirurgie Mini-Invasive du Pied, GRECMIP, 4Foot and Ankle Unit, Hospital Quirón Barcelona, 5Foot and Ankle Unit, Orthopedic and Trauma Surgery, Royal London Hospital, Barts Health NHS Trust, 6Department of Orthopedic Surgery, Foot and Ankle Unit, University of Minnesota Minimally invasive surgical (MIS) procedures rely on anatomical references to localize structures not directly visible to the surgeon. This manuscript describes a combined method of plane-by-plane dissection and sectional anatomy of fresh-frozen specimens to locate the structures at risk during MIS procedures. Bioengineering Preparation, Purification, and Use of Fatty Acid-containing Liposomes Lin Jin*1,2, Aaron E. Engelhart*1,3, Katarzyna P. Adamala1,3, Jack W. Szostak1 1Howard Hughes Medical Institute and Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, 2Department of Biomedical Engineering, Boston University, 3Department of Genetics, Cell Biology, and Development, University of Minnesota Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles. Bioengineering Use of a Rat Model to Study Ventral Abdominal Hernia Repair Mark A. Suckow1, Felicia D. Duke Boynton2, Chad Johnson3 1Department of Veterinary Population Medicine, University of Minnesota, 2Research Animal Resources, University of Minnesota, 3Cook Biotech, Inc. This manuscript describes methods associated with creation and repair of a ventral abdominal wall defect (hernia). This model can be used to study repair strategies such as those that use implanted materials. In this manuscript, repair of the experimental hernia with porcine small intestinal submucosa is presented as an example. Cancer Research A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s Erik S. Carlson1,2, Pramod Upadhyaya2, Stephen S. Hecht2 1Department of Pharmacology, University of Minnesota, 2Masonic Cancer Center, University of Minnesota α-hydroxylation of carcinogenic nitrosamines by cytochrome P450s is the accepted metabolic pathway that produces DNA-damaging intermediates, which cause mutations. However, new data indicates further oxidation to nitrosamides can occur. We describe a general method for detecting nitrosamides produced from in vitro cytochrome P450-catalyzed metabolism of nitrosamines. Immunology and Infection In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues Shengbin Li1, Gwantwa Mwakalundwa1, Pamela J. Skinner1 1Department of Veterinary and Biomedical Sciences, University of Minnesota Here, we describe a method that combines in situ MHC-tetramer staining with immunohistochemistry to determine localization, phenotype, and quantity of antigen-specific T cells in tissues. This protocol is used to determine the spatial and phenotypic characteristics of antigen-specific CD8 T cells relative to other cell type and structures in tissues. Neuroscience Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters Kelsey M. Moore1, Brett T Himmler1, Benjamin A Teplitzky2, Matthew D Johnson2,3, Robert L Meisel1 1Department of Neuroscience, University of Minnesota, 2Department of Biomedical Engineering, University of Minnesota, 3Institute for Translational Neuroscience, University of Minnesota This paper details the use of fixed-potential amperometric recordings using carbon fiber electrodes and enzymatic biosensor technology to measure the release of dopamine and glutamate with high temporal resolution during natural rewarding behavior in the female hamster. Bioengineering Ammonia Synthesis at Low Pressure Edward Cussler1, Alon McCormick1, Michael Reese2, Mahdi Malmali1 1Department of Chemical Engineering and Materials Science, University of Minnesota – Twin Cities, 2West Central Research and Outreach Center, University of Minnesota – Morris Ammonia can be synthesized at low pressure by using a conventional catalyst and an ammonia selective absorbent. Bioengineering Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System Mark Rustad1, Allen Eastlund2, Ryan Marshall1, Paul Jardine2, Vincent Noireaux1 1School of Physics and Astronomy, University of Minnesota, 2Department of Diagnostic and Biological Sciences and Institute for Molecular Virology, University of Minnesota A new generation of cell-free transcription-translation platforms has been engineered to construct biochemical systems in vitro through the execution of gene circuits. In this article, we describe how bacteriophages, such as MS2, ΦΧ174, and T7, are synthesized from their genome using an all E. coli cell-free TXTL system. Cancer Research Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis Charles E. Gast1, Aubie K. Shaw1,2, Melissa H. Wong1,3, Lisa M. Coussens1,3 1Cell, Developmental & Cancer Biology, Oregon Health & Science University, 2University of Minnesota, 3Knight Cancer Institute, Oregon Health & Science University Pre-clinical models evaluating adjuvant therapy targeting breast cancer metastasis are lacking. To address this, we developed a murine model of de novo pulmonary mammary adenocarcinoma metastasis, wherein therapies administered in the adjuvant setting (post surgical resection of primary tumors) can be evaluated for efficacy in impacting previously seeded pulmonary metastases. Genetics Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing Marisa E. Miller*1,2, Katie L. Liberatore*1,3, Shahryar F. Kianian1,3 1Cereal Disease Laboratory, United States Department of Agriculture-Agricultural Research Service, 2Department of Horticultural Science, University of Minnesota, 3Department of Plant Pathology, University of Minnesota The comparison and optimization of two plant organellar DNA enrichment methods are presented: traditional differential centrifugation and fractionation of the total gDNA based on methylation status. We assess the resulting DNA quantity and quality, demonstrate performance in short-read next-generation sequencing, and discuss the potential for use in long-read single-molecule sequencing. Environment A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples Lawrence G. Oates1, Harry W. Read2, Jessica L. M. Gutknecht3, David S. Duncan1, Teri B. Balser4, Randall D. Jackson1 1Department of Agronomy and Great Lakes Bioenergy Research Center, University of Wisconsin - Madison, 2Department of Soil Science, University of Wisconsin - Madison, 3Department of Soil, Water, and Climate, University of Minnesota, 4Faculty of Science and Engineering, Curtin University The article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples. Developmental Biology Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles Abhijit Nirwane1, Jyoti Gautam1, Yao Yao1 1College of Pharmacy, University of Minnesota This work describes a FACS-based protocol that allows for easy and simultaneous isolation of type I and type II pericytes from skeletal muscles. Neuroscience Fiber Connections of the Supplementary Motor Area Revisited: Methodology of Fiber Dissection, DTI, and Three Dimensional Documentation Baran Bozkurt1, Kaan Yagmurlu2, Erik H. Middlebrooks3, Zuzan Cayci4, Orhun M. Cevik1, Ali Karadag5, Sean Moen1, Necmettin Tanriover6, Andrew W. Grande1 1Department of Neurosurgery, University of Minnesota, 2Department of Neurosurgery, Barrow Neurological Institute, St. Josephs Hospital and Medical Center, 3Department of Radiology, University of Alabama at Birmingham, 4Department of Radiology, University of Minnesota, 5Department of Neurosurgery, Tepecik Training and Research Hospital, 6Department of Neurosurgery, Cerrahpasa Medical School, University of Istanbul The purpose of this study is to show each step of the fiber dissection technique on human cadaveric brains, the 3D documentation of these dissections, and the diffusion tensor imaging of the anatomically dissected fiber pathways. Developmental Biology Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish Elaine L. Welch*1, Celeste C. Eno*1, Sreelaja Nair2, Robin E. Lindeman3, Francisco Pelegri1 1Laboratory of Genetics, University of Wisconsin-Madison, 2Department of Biological Sciences, Tata Institute of Fundamental Research, 3Department of Genetics, Cell Biology, and Development, University of Minnesota An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here. Cancer Research A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies Katherine A. Murphy*1,2, Britnie R. James*1,2,3, Andrew Wilber4,5, Thomas S. Griffith1,2,3 1Department of Urology, University of Minnesota, 2Masonic Cancer Center, University of Minnesota, 3Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota, 4Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, 5Simmons Cancer Institute Implementation of an orthotopic model of renal cell carcinoma in immunocompetent mice affords the investigator a clinically-relevant system defined by the presence of a primary renal tumor and lung metastases in the same animal. This system can be used to preclinically test a variety of treatments in vivo. Engineering Fabrication of Nanopillar-Based Split Ring Resonators for Displacement Current Mediated Resonances in Terahertz Metamaterials Chao Liu1, Joseph Schauff1, Seokhyeong Lee1, Jeong-Hyun Cho1 1Department of Electrical and Computer Engineering, University of Minnesota A protocol for the design and fabrication of a novel nanopillar-based split ring resonator (SRR) is presented. Genetics Generation of Fluorescent Protein Fusions in Candida Species Sara Gonia1, Judith Berman2, Cheryl A. Gale1 1Department of Pediatrics, University of Minnesota, 2Department of Molecular Microbiology and Biotechnology, Tel Aviv University PCR-mediated gene modification can be used to generate fluorescent protein fusions in Candida species, which facilitates visualization and quantitation of yeast cells and proteins. Herein, we present a strategy for constructing a fluorescent protein fusion (Eno1-FP) in Candida parapsilosis. Biochemistry Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution Gatikrushna Singh*1, Sarah M. Fritz*2, Arnaz Ranji2, Deepali Singh3, Kathleen Boris-Lawrie1,2 1Department of Veterinary & Biomedical Sciences, University of Minnesota, 2Department of Veterinary Biosciences, Ohio State University, 3School of Biotechnology, Gautam Buddha University This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A. Bioengineering Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies Mary G. Garry1, Stefan M. Kren1, Daniel J. Garry1 1Lillehei Heart Institute, University of Minnesota In these studies, we provide methodology for novel, neonatal, murine cardiac scaffolds for use in regenerative studies. Neuroscience Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity Cayla M. Duffy1,2, Shihab Ahmed1, Ce Yuan1,2,3, Vijayakumar Mavanji1, Joshua P. Nixon1,2, Tammy Butterick1,2,4 1Minneapolis Veterans Affairs Health Care System, 2Department of Food Science and Nutrition, University of Minnesota, 3Biomedical Informatics and Computational Biology Program, University of Minnesota, 4Minnesota Obesity Center, University of Minnesota Microglia (immune cells of the brain), are used as a surrogate biosensor to determine how nanoparticles influence neurotoxicity. We describe a series of experiments designed to assay microglial response to nanoparticles and exposure of hypothalamic neurons to supernatant from activated microglia to determine neurotoxicity. Bioengineering Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids Bradley P. Weegman1, Ahmad Essawy2, Peter Nash2, Alexandra L. Carlson2, Kristin J. Voltzke3, Zhaohui Geng2, Marjan Jahani2, Benjamin B. Becker2, Klearchos K. Papas4, Meri T. Firpo2 1Radiology, University of Minnesota, 2Medicine, University of Minnesota, 3School of Public Health, University of Minnesota, 4Surgery, University of Arizona Nutrient regulation using continuous growth adjusted feeding improves growth rates of mammalian cell spheroids compared to intermittent batch feeding for cultures in stirred suspension bioreactors. This study demonstrates the methods required for establishing simple adjusted rate fed cultures. Neuroscience Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna Shala J. Hankison1, Meredith S. Palmer2 1Department of Zoology, Ohio Wesleyan University, 2Department of Ecology, Evolution, & Behavior, University of Minnesota This protocol outlines the use of spectrophotometry to detect ultraviolet-reflecting structures on organisms (in this example, the sailfin molly Poecilia latipinna) and describes dichotomous choice tests for fish that allow inferences to be made on the role of ultraviolet cues during mate selection. Biology G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay Ansley Semack1, Rabia U. Malik2, Sivaraj Sivaramakrishnan1 1Genetics, Cell Biology, and Development, University of Minnesota, 2Department of Cell and Developmental Biology, University of Michigan Simple methods to detect the selective activation of G proteins by G protein-coupled receptors remain an outstanding challenge in cell signaling. Here, Fӧrster resonance energy transfer (FRET) biosensors have been developed by pairwise tethering a GPCR to G protein peptides to probe conformational changes at controlled concentrations in live cells. Biology Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System Emanuel G. Worst1, Matthias P. Exner2, Alessandro De Simone2, Marc Schenkelberger1, Vincent Noireaux3, Nediljko Budisa2, Albrecht Ott1 1Department of Experimental Physics, Saarland University, 2Institute of Chemistry, Technische Universität Berlin, 3School of Physics and Astronomy, University of Minnesota An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies. Medicine Testing the Efficacy of Pharmacological Agents in a Pericardial Target Delivery Model in the Swine Tinen L. Iles1, Brian Howard2, Stephen Howard3, Stephen Quallich2, Christopher Rolfes2, Eric Richardson4, Hanna R. Iaizzo5, Paul A. Iaizzo1 1Surgery, University of Minnesota, 2Biomedical Engineering, University of Minnesota, 3Medtronic, Inc., 4Bioengineering, Rice University, 5Pharmacy, University of Wisconsin We have developed a swine model for the target delivery of pharmacological agents within the pericardial space/fluid. Using this approach, the relative benefits of administered agents on induced atrial fibrillation, relative refractory periods and/or ischemic protection can be investigated. Biology Measuring Pressure Volume Loops in the Mouse DeWayne Townsend1 1Department of Integrative Biology and Physiology, University of Minnesota This manuscript describes a detailed protocol for the collection of pressure-volume data from the mouse. Immunology and Infection Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation Charles J. Benck1, Tijana Martinov2, Brian T. Fife2, Devavani Chatterjea1 1Department of Biology, Macalester College, 2Department of Medicine, Division of Rheumatic and Autoimmune Diseases, Center for Immunology, University of Minnesota This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols. Medicine A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting Eric B. Berens1, Jon M. Holy2, Anna T. Riegel1, Anton Wellstein1 1Lombardi Comprehensive Cancer Center, Department of Oncology, Georgetown University, 2Department of Biomedical Sciences, University of Minnesota This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored. Environment Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies Petra Kranzfelder1, Alyssa M. Anderson2, Alexander T. Egan1, Jane E. Mazack1, R. William Bouchard, Jr.3, Moriya M. Rufer4, Leonard C. Ferrington, Jr.1 1Department of Entomology, University of Minnesota, 2Biology, Chemistry & Physics, and Mathematics Department, Northern State University, 3Environmental Analysis and Outcomes Division, Minnesota Pollution Control Agency, 4RMB Environmental Laboratories, Inc. Rapid bioassessment protocols using benthic macroinvertebrates are often used to monitor and assess water quality. An efficient protocol involves collections of Chironomidae surface-floating pupal exuviae (SFPE). Here, techniques for field collection, laboratory processing, slide mounting, and identification of Chironomidae SFPE are described. Bioengineering Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films Eric S. Hald1, Kerianne E. Steucke1, Jack A. Reeves1, Zaw Win1, Patrick W. Alford1 1Department of Biomedical Engineering, University of Minnesota We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses. Behavior Morris Water Maze Test: Optimization for Mouse Strain and Testing Environment Daniel S. Weitzner1, Elizabeth B. Engler-Chiurazzi2, Linda A. Kotilinek3, Karen Hsiao Ashe3,4,5, Miranda Nicole Reed1,6 1Department of Psychology, Behavioral Neuroscience, West Virginia University, 2Department of Physiology and Pharmacology, West Virginia University, 3Department of Neurology, N. Bud Grossman Center for Memory Research and Care, University of Minnesota, 4Department of Neuroscience, N. Bud Grossman Center for Memory Research and Care, University of Minnesota, 5GRECC, VA Medical Center, 6Center for Neuroscience, Center for Basic and Translational Stroke Research, West Virginia University This manuscript describes a Morris water maze (MWM) protocol tailored for use with a commonly used mouse model of Alzheimer's disease. The MWM is widely used in transgenic mouse models. Implementation of a procedure sensitive to the background strain of the mouse model is essential for detecting group differences. Biology Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples Percy Griffin1,2, Ashley Sexton3, Lauren Macneill3, Yoshie Iizuka3,4, Michael K. Lee1,2, Martina Bazzaro3,4 1Department of Neuroscience, University of Minnesota, 2Institute for Translational Neuroscience, University of Minnesota, 3Department of Obstetrics, Gynecology, and Women’s Heath, University of Minnesota, 4Masonic Cancer Center, University of Minnesota The current protocol details a method for measuring the activity of functionally homologous deubiquitinating enzymes. Specialized probes covalently modify the enzyme and allow for detection. This method holds the potential to identify new therapeutic targets. Immunology and Infection High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization Jana Ninković1,3, Sabita Roy1,2 1Department of Pharmacology, University of Minnesota, 2Department of Surgery, University of Minnesota, 33M Corporate Research Laboratory Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization. Neuroscience The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism Sara Tremblay1, Vincent Beaulé1, Sébastien Proulx2, Louis-Philippe Lafleur1, Julien Doyon1, Małgorzata Marjańska3, Hugo Théoret1 1Department of Psychology, University of Montréal, 2Montreal Neurological Institute, McGill University, 3Center for Magnetic Resonance Research and Department of Radiology, University of Minnesota This article aims to describe a basic protocol for combining transcranial direct current stimulation (tDCS) with proton magnetic resonance spectroscopy (1H-MRS) measurements to investigate the effects of bilateral stimulation on primary motor cortex metabolism. Bioengineering CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells Jing Ge*1, Somsak Prasongtanakij*2, David K. Wood3, David M. Weingeist1, Jessica Fessler1, Panida Navasummrit2, Mathuros Ruchirawat2, Bevin P. Engelward1 1Department of Biological Engineering, Massachusetts Institute of Technology, 2Environmental Toxicology, Chulabhorn Graduate Institute, 3Department of Biomedical Engineering, University of Minnesota We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics. Immunology and Infection Isolation, Identification, and Purification of Murine Thymic Epithelial Cells Yan Xing1, Kristin A. Hogquist1 1Department of Laboratory Medicine & Pathology, Center for Immunology, University of Minnesota Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution. Medicine Shrinkage of Dental Composite in Simulated Cavity Measured with Digital Image Correlation Jianying Li1, Preetanjali Thakur1, Alex S. L. Fok1 1Minnesota Dental Research Center for Biomaterials and Biomechanics, School of Dentistry, University of Minnesota In order to understand the spatial development of polymerization shrinkage stress in dental resin-composite restorations, Digital Image Correlation was used to provide full-field displacement/strain measurement of restored model glass cavities by correlating images of the restoration taken before and after polymerization. Bioengineering Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method Nathan J. Wittenberg1, Timothy W. Johnson1, Luke R. Jordan2, Xiaohua Xu3, Arthur E. Warrington3, Moses Rodriguez3,4, Sang-Hyun Oh1,2 1Department of Electrical and Computer Engineering, University of Minnesota, 2Department of Biomedical Engineering, University of Minnesota, 3Department of Neurology, Mayo Clinic College of Medicine, 4Department of Immunology, Mayo Clinic College of Medicine Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles. Neuroscience Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model Tammy A. Butterick1,2, Cayla M. Duffy1,2, Rachel E. Lee3, Charles J. Billington1,2,4, Catherine M. Kotz1,2, Joshua P. Nixon1,2 1Department of Veterans Affairs, Minneapolis Veterans Affairs Health Care System, 2Department of Food Science and Nutrition, University of Minnesota, 3Department of Integrative Biology and Physiology, University of Minnesota, 4Department of Medicine, University of Minnesota Medical School, University of Minnesota Multiplex assays can provide beneficial information for basic cellular mechanisms and eliminate waste of reagents and unnecessary repetitive experiments. We describe here a multiplex caspase-3/7 activity assay, using fluorescent- and luminescent-based methods, to determine cell viability in an in vitro hypothalamic model following oxidative challenge with palmitic acid. Biology Mouse Genome Engineering Using Designer Nucleases Mario Hermann1, Tomas Cermak2, Daniel F. Voytas2, Pawel Pelczar1 1Institute of Laboratory Animal Science, University of Zurich, 2Department of Genetics, Cell Biology & Development and Center for Genome Engineering, University of Minnesota Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications. Medicine Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens Lee J. Pribyl1, Kathleen A. Coughlin1, Thanasak Sueblinvong1, Kristin Shields2, Yoshie Iizuka1,3, Levi S. Downs1,3, Rahel G. Ghebre1,3, Martina Bazzaro1,3 1Department of Obstetrics, Gynecology, and Women's Heath, University of Minnesota, 2Department of Obstetrics and Gynecology, Maricopa Medical Center and St Josephs Hospital and Medical Center, 3Masonic Cancer Center, University of Minnesota This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications. Biology Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology Zachary Z. Sun*1, Clarmyra A. Hayes*2, Jonghyeon Shin3, Filippo Caschera4, Richard M. Murray2, Vincent Noireaux4 1Department of Biology, California Institute of Technology, 2Department of Bioengineering, California Institute of Technology, 3Synthetic Biology Center, Department of Bioengineering, Massachusetts Institute of Technology, 4School of Physics and Astronomy, University of Minnesota This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data. Bioengineering Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs) Allison M. Bock*1,2, David Knorr*1,2, Dan S. Kaufman1,2 1Department of Medicine (Hematology, Oncology, and Transplant), University of Minnesota, Minneapolis, 2Stem Cell Institute, University of Minnesota, Minneapolis This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs. Bioengineering Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens Julianne Spencer1,2, Emily Fitch3, Paul A. Iaizzo1,2,4,5 1Department of Surgery, University of Minnesota, 2Department of Biomedical Engineering, University of Minnesota, 3Department of Biology, University of Minnesota, 4Department of Integrative Biology & Physiology, University of Minnesota, 5Institute for Engineering in Medicine, University of Minnesota The objective of this research is to recreate and then access the anatomy of the human cardiac venous system using 3D reconstructions generated from contrast-computed tomography scans. Medicine Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR Callie L. Janik1,2, Timothy K. Starr1,2 1Department of Obstetrics, Gynecology & Women's Health, Masonic Cancer Center, University of Minnesota, Minneapolis, 2Department of Genetics, Cell Biology & Development, Center for Genome Engineering, University of Minnesota, Minneapolis A method of identifying unknown drivers of carcinogenesis using an unbiased approach is described. The method uses the Sleeping Beauty transposon as a random mutagen directed to specific tissues. Genomic mapping of transposon insertions that drive tumor formation identifies novel oncogenes and tumor suppressor genes Neuroscience Creating Objects and Object Categories for Studying Perception and Perceptual Learning Karin Hauffen1,2,3, Eugene Bart4, Mark Brady5, Daniel Kersten6, Jay Hegdé1,2,3 1Brain and Behavior Discovery Institute, Georgia Health Sciences University, 2Vision Discovery Institute, Georgia Health Sciences University, 3Department of Opthalmology, Georgia Health Sciences University, 4Intelligent Systems Laboratory, Palo Alto Research Center, 5Pattern Recognition Systems, Palo Alto Research Center, 6Department of Psychology, University of Minnesota We describe a novel methodology for creating naturalistic 3-D objects and object categories with precisely defined feature variations. We use simulations of the biological processes of morphogenesis and phylogenesis to create novel, naturalistic virtual 3-D objects and object categories that can then be rendered as visual images or haptic objects. Biology Performing Custom MicroRNA Microarray Experiments Xiaoxiao Zhang1, Yan Zeng1,2 1Department of Pharmacology, University of Minnesota, 2Masonic Cancer Center, University of Minnesota A simple procedure of performing custom microRNA microarray experiments is described. The steps include isolating RNA, labeling RNA and reference DNA, hybridizing the samples to microarrays, scanning the microarrays, quantifying and analyzing hybridization signals. Biology Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica John D. Chan1, Jonathan S. Marchant1 1Department of Pharmacology and The Stem Cell Institute, University of Minnesota Medical School An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article. Immunology and Infection Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field John Y. Takekawa1, Nichola J. Hill1,2, Annie K. Schultz1, Samuel A. Iverson1, Carol J. Cardona3,4, Walter M. Boyce2, Joseph P. Dudley5 1USGS Western Ecological Research Center, 2Wildlife Health Center, University of California, Davis, 3Department of Population Health and Reproduction, University of California, Davis, 4Department of Veterinary and Biomedical Sciences, University of Minnesota, 5Science Applications International Corporation This study describes diagnosis of avian influenza in wild birds using a portable rRT-PCR system. The method takes advantage of freeze-dried reagents to screen wild birds in a non-laboratory setting, typical of an outbreak scenario. Use of molecular tools provides accurate and sensitive alternatives for rapid diagnosis. Neuroscience Labeling F-actin Barbed Ends with Rhodamine-actin in Permeabilized Neuronal Growth Cones Bonnie M. Marsick1, Paul C. Letourneau1 1Department of Neuroscience, University of Minnesota A method to visualize and quantify F-actin barbed ends in neuronal growth cones is described. After culturing neurons on glass coverslips, cells are permeabilized with a saponin-containing solution. Then, a short incubation with the saponin buffer containing rhodamine-actin incorporates fluorescent actin onto free actin barbed ends. Biology Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi Jonathan S. Schilling1, K. Brook Jacobson*1 1Department of Bioproducts and Biosystems Engineering, University of Minnesota This video demonstrates a controlled environment approach to study degradation of lignocellulosic plant tissues by aerobic fungi. The ability to control nutrient sources and moisture is a key advantage of agar-block microcosms, but the approach often yields mixed success. We address critical pitfalls to yield reproducible, low-variability results. Neuroscience An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells Tiffany M Schmidt1, Paulo Kofuji1 1Department of Neuroscience, University of Minnesota This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology. Biology A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ Qingsheng Li1, Pamela J. Skinner2, Lijie Duan1, Ashley T. Haase1 1Department of Microbiology, Medical School, University of Minnesota, 2Department of Veterinary and Biomedical Sciences, University of Minnesota A technique combining in situ tetramer staining and in situ hybridization (ISTH) enables visualization, mapping and analysis of the spatial proximity of virus-specific CD8+ T cells to their virus-infected targets, and determination of the quantitative relationships between these immune effectors and targets to infection outcomes. Biology Gibberella zeae Ascospore Production and Collection for Microarray Experiments. Matias Pasquali1,2, Corby Kistler3 1Cereal Disease Laboratory, USDA, 2University of Minnesota/ Agroinnova, University of Torino, 3Cereal Disease Laboratory, University of Minnesota To study the developmental processes of ascospores in Gibberella zeae, a procedure for collection under sterile conditions is filmed in order to generate the highest level of information for protocol description. This should facilitate the reproducibility of the experiment, a crucial aspect when full genome expression profile tests are implemented.
