University of Colorado School of Medicine, Anschutz Medical Campus

3 ARTICLES PUBLISHED IN JoVE

Developmental Biology

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Patrick S. McGrath^1,2,3, Nicole Diette^1,2, Igor Kogut^1,2,3, Ganna Bilousova^1,2,3

¹Department of Dermatology, University of Colorado School of Medicine, Anschutz Medical Campus, ²Charles C. Gates Center for Regenerative Medicine, University of Colorado School of Medicine, Anschutz Medical Campus, ³Stem Cell Biobank and Disease Modeling Core, University of Colorado School of Medicine, Anschutz Medical Campus

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

Immunology and Infection

Measuring Mitochondrial Function of Naïve and Effector CD8 T Cells

Jacqueline A. Turner^1,2, Elizabeth Katsnelson³, Raul M. Torres¹

¹Department of Immunology and Microbiology, University of Colorado School of Medicine, Anschutz Medical Campus, ²Medical Scientist Training Program, University of Colorado School of Medicine, Anschutz Medical Campus, ³Division of Surgical Oncology, Department of Surgery, University of Colorado School of Medicine, Anschutz Medical Campus

CD8 T cell bioenergetics can be interrogated using the Mito Stress Test. This methodology can be used to study acute and chronic metabolic programming. This protocol describes approaches to examine the relationships between T cell receptor biology and bioenergetic analysis.

Genetics

CIRCLE-Seq for Interrogation of Off-Target Gene Editing

Jeffrey Inen^1,2, Chann Makara Han^1,2, David M. Farrel³, Ganna Bilousova^1,2, Igor Kogut^1,2

¹Department of Dermatology, University of Colorado School of Medicine, Anschutz Medical Campus, ²Gates Institute, University of Colorado School of Medicine, Anschutz Medical Campus, ³Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Anschutz Medical Campus

A significant barrier to technologies like CRISPR is the off-target events that can disrupt vital genes. 'Circularization for In Vitro Reporting of Cleavage Effects by Sequencing' (CIRCLE-seq) is a technique designed to identify unintended cleavage sites. This method maps the genome-wide activity of CRISPR-Cas9 with high sensitivity and without bias.