Accedi

National Institute On Deafness And Other Communication Disorders

2 ARTICLES PUBLISHED IN JoVE

Developmental Biology

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Khujista D. Haque¹, Atul K. Pandey¹, Matthew W. Kelley², Chandrakala Puligilla¹

¹Department of Pathology and Laboratory Medicine, Medical University of South Carolina, College of Medicine, ²Laboratory of Cochlear Development, NIDCD, NIH

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Neuroscience

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Kaya J.E. Matson¹, Anupama Sathyamurthy¹, Kory R. Johnson², Michael C. Kelly^3,4, Matthew W. Kelley³, Ariel J. Levine¹

¹Spinal Circuits and Plasticity Unit, National Institute of Neurological Disorders and Stroke, ²Bioinformatics Section, Information Technology Program, National Institute of Neurological Disorders and Stroke, ³Laboratory of Cochlear Development, National Institute on Deafness and Other Communication Disorders, ⁴Single Cell Analysis Facility, Frederick National Laboratory

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.