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7 ARTICLES PUBLISHED IN JoVE

Biology

Gibberella zeae Ascospore Production and Collection for Microarray Experiments.

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.

Biology

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Matias Pasquali¹, Frédéric Giraud¹, Jean Paul Lasserre¹, Sebastien Planchon¹, Lucien Hoffmann¹, Torsten Bohn¹, Jenny Renaut¹

¹Department of Environment and Agro-Biotechnologies (EVA), Nutrition and Toxicology Unit (NuTox), Centre de Recherche Public-Gabriel Lippmann

Protein extraction for proteomic analyses in fungal species requires high levels of standardization to be accomplished according with the minimum information about a proteomic experiment (MIAPE) guidelines. We present a video-protocol that includes a procedure for minimizing experimental bias during toxin induction and protein extraction from Fusarium spp.

Immunology and Infection

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Justin W Fischer¹, Tracy A Nichols¹, Gregory E Phillips¹, Kurt C VerCauteren¹

¹Animal and Plant Health Inspection Service, Wildlife Services, National Wildlife Research Center, USDA

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

Biology

Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model

Pablo Bermejo-Alvarez^1,2, Ki-Eun Park^1,2, Bhanu P. Telugu^1,2

¹Animal Bioscience and Biotechnology Laboratory, United States Department of Agriculture, ²Department of Animal and Avian Sciences, University of Maryland

Utero-tubal embryo transfer uses the utero-tubal junction as a barrier to prevent the embryo outflow that may occur when performing uterine transfer. Vasectomized males are required to obtain pseudopregnant recipients for embryo transfer. Both techniques are discussed.

Biology

Visualizing Stromule Frequency with Fluorescence Microscopy

Jacob O. Brunkard¹, Anne M. Runkel², Patricia Zambryski²

¹Plant Gene Expression Center, Agricultural Research Service, USDA, ²Department of Plant and Microbial Biology, University of California, Berkeley

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

Bioengineering

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Anjali Nandal^1,2, Barbara Mallon³, Bhanu P. Telugu^1,2,4

¹Department of Animal and Avian Sciences, University of Maryland, ²Animal Bioscience and Biotechnology Laboratory, ARS, USDA, ³NIH Stem Cell Unit, Bethesda, National Institutes of Health, ⁴RenOVAte Biosciences Inc

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

Biology

Sieving Fruit Pulp to Detect Immature Tephritid Fruit Flies in the Field

Amy L. Roda¹, Gary Steck², Thomas Fezza³, Todd Shelly³, Rita Duncan⁴, Nicholas Manoukis⁵, Lori Carvalho⁵, Abbie Fox⁶, Paul Kendra⁷, Daniel Carrillo⁴

¹Animal Plant Health Inspection Service (APHIS), Plant Protection and Quarantine (PPQ), Science and Technology (S&T) Miami, United States Department of Agriculture (USDA), ²Division of Plant Industry, Florida Department of Agriculture and Consumer Services, ³APHIS, PPQ, S&T, USDA, ⁴IFAS, Tropical Research and Education Center, University of Florida, ⁵Agricultural Research Service (ARS), Pacific Basin Agricultural Research Center, USDA, ⁶APHIS, PPQ, Field Operations, USDA, ⁷ARS, Subtropical Horticulture Research Station, USDA

Increasing the detection of immature tephritid fruit flies in the field can trigger timely efforts to eliminate populations of these destructive pests. Detecting late instar larvae is faster and more accurate when mushing host fruit in a bag and passing the pulp through a series of sieves than hand cutting and visual inspection.