NG2 Knock-Out in Neu7 and A7 Astrocyte Cell Lines to Promote Neuronal Regeneration — Presentation Detail — Celebration of Scholars

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Additional Information

More information is available at carthage.edu/celebration-scholars/. The following are members of the Research, Scholarship, and Creativity Committee who are eager to listen to ideas and answer questions:

Jun Wang
Kim Instenes
John Kirk
Nora Nickels
Andrew Pustina
James Ripley

NG2 Knock-Out in Neu7 and A7 Astrocyte Cell Lines to Promote Neuronal Regeneration

Name: Arielle Hay
Major: Neuroscience, Biology
Hometown: Saint Paul, MN
Faculty Sponsor:
Other Sponsors:
Type of research: Independent research

Abstract

Stroke is one of the leading causes of death in America and worldwide. After a stroke, neuronal regeneration is inhibited due to the astrocytes surrounding the tissue becoming reactive. Reactive astrocytes are non-permissive to axon outgrowth, and improving our understanding of astrocyte reactivity can facilitate development of more targeted therapies for neuronal regeneration. Neu7 cells are an astrocyte cell line that models this reactive phenotype due to an increased production of inhibitory chondroitin sulfate proteoglycans. Of these proteoglycans, chondroitin sulfate proteoglycan 4 (NG2) has been found to be the most inhibitory to axon regeneration. A7 cells are a model cell line for astrocytes that are permissive to axon outgrowth, having a downregulated production of NG2. In our study, the CRISPR/Cas9 system was used to produce NG2 knockout Neu7 and A7 cell lines. NG2 is a 35,046 base pair long gene with ten exons. We created guide sequences targeting exons 1, 3, 5 and 10, and bacterial cloning was used to construct plasmids containing guide sequences, Cas9 and green fluorescent protein (GFP). Sanger sequencing and alignment identified correctly constructed plasmids. Transfection efficiency was determined for select plasmids by transfection into Neu7 cells. Ongoing experiments will continue to determine optimal conditions for transfection and develop neuronal co-cultures with transfected Neu7 and A7 cells. We believe these experiments will aid in the development of new therapies for neuronal regeneration.

Poster file