Undergraduate Honors Theses

Thesis Defended

Spring 2015

Document Type

Thesis

Type of Thesis

Departmental Honors

Department

Molecular, Cellular, & Developmental Biology

First Advisor

Bradley Olwin

Second Advisor

Jennifer Martin

Third Advisor

Benjamin Teitelbaum

Abstract

Isolation of myogenic cell populations from whole skeletal muscle is necessary to elucidate the molecular repair mechanisms involved in skeletal muscle maintenance and regeneration that can be damaged when affected by diseases or aging, often severely impacting quality of life and lifespan. Skeletal muscle tissue is composed of hundreds of myofibers that are syncytia comprised of hundreds of nuclei named myonuclei. External to the sarcolemma(plasma membrane) of each myofiber is the basal lamina. Satellite cells, which reside between the basal lamina and the sarcolemma, are self-renewing muscle stem cells that maintain and repair muscle tissue. I have developed a method to purify myonuclei and satellite cell nuclei from non-myogenic nuclei in muscle to investigate molecular differences among nuclei present in muscle.. I compared multiple methods of cellular lysis and homogenization to separate nuclei from the whole muscle tissue. Next, I tested two methods for nuclear isolation and found that a percoll gradient efficiently removed cellular debris from the nuclei. Finally, I stained for Pax7 and Myogenin, which are specific for satellite cells and myonuclei respectively. I found that percoll isolation effectively isolated 45% of myogenic nuclei from whole muscle tissue. A protocol for the isolation of myogenic nuclei will allow for nuclear sorting of myonuclei and satellite cell nuclei from non-myogenic nuclei, which has been previously difficult to achieve. Effective isolation and sorting of nuclei away from the intracellular environment without cell culture will allow for investigation of diseases that stem from errors in myogenic nuclei.

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