Cincinnati Children's Hospital

6 ARTICLES PUBLISHED IN JoVE

Bioengineering

Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

Yoonsuck Choe¹, David Mayerich², Jaerock Kwon³, Daniel E. Miller¹, Chul Sung¹, Ji Ryang Chung¹, Todd Huffman⁴, John Keyser¹, Louise C. Abbott⁵

¹Department of Computer Science and Engineering, Texas A&M University, ²Beckman Institute for Advanced Science and Technology, University of Illinois, ³Department of Electrical and Computer Engineering, Kettering University, ⁴3Scan, ⁵Department of Veterinary Integrative Biosciences, Texas A&M University

The full process from brain specimen preparation to serial sectioning imaging using the Knife-Edge Scanning Microscope, to data visualization and analysis is described. This technique is currently used to acquire mouse brain data, but it is applicable to other organs, other species.

Medicine

3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

Teagan J. Walter^1,2, Erin E. Sparks³, Stacey S. Huppert²

¹Department of Cell and Developmental Biology, Center for Stem Cell Biology, Vanderbilt University, ²Division of Gastroenterology, Hepatology, and Nutrition, Cincinnati Children's Hospital, ³Department of Biology, Duke University

A method of visualizing and quantifying the 3-dimensional structure of mouse hepatic portal vein or intrahepatic bile duct is described. This resin cast technique can also be applied to other ductal or vascular systems and allows for in situ visualization or quantification of a system's intact communicating architecture.

JoVE Journal

Reconstitution Of β-catenin Degradation In Xenopus Egg Extract

Tony W. Chen^*1, Matthew R. Broadus^*1, Stacey S. Huppert², Ethan Lee^1,3

¹Department of Cell and Developmental Biology and Program in Developmental Biology, Vanderbilt University Medical Center, ²Division of Gastroenterology, Hepatology & Nutrition and Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, ³Vanderbilt Ingram Cancer Center, Vanderbilt University School of Medicine

A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.

Biology

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Sheldon G. Watts^*1, Justin J. Crowder^*1, Samuel Z. Coffey^1,2, Eric M. Rubenstein¹

¹Department of Biology, Ball State University, ²Division of Nephrology, Cincinnati Children's Hospital

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

Developmental Biology

Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells

Ashley Ulm¹, Christopher N. Mayhew², Jason Debley³, Gurjit K. Khurana Hershey⁴, Hong Ji^1,4

¹Pyrosequencing Core, Cincinnati Children's Hospital, ²Division of Developmental Biology, Cincinnati Children's Hospital, ³Division of Pulmonary Medicine, Seattle Children's Hospital, ⁴Division of Asthma Research, Cincinnati Children's Hospital

This publication demonstrates methods for successful sampling and culture of nasal epithelial mucosa from children, and reprogramming these cells to induced Pluripotent Stem Cells (iPSCs).

Biology

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

Daniel E. Miller¹, Zubin H. Patel^1,2,3, Xiaoming Lu^1,3, Arthur T. Lynch¹, Matthew T. Weirauch^*1,4, Leah C. Kottyan^*1

¹Center for Autoimmune Genomics and Etiology, Cincinnati Children's Hospital, ²Medical Scientist Training Program, University of Cincinnati, ³Immunology Graduate Program, University of Cincinnati, ⁴Divisions of Biomedical Informatics and Developmental Biology, Cincinnati Children's Hospital

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.