Jesus Trujillo

Phenotypic Analysis of the Actbcg/Actg1-/- Cell Model

In mammals, six unique actin isoforms are expressed in a tissue-specific manner. The cytoplasmic isoforms, 𝛽- (Actb) and 𝛾- (Actg1) actin, share 98.9% homology in the amino acid sequence but function differently. Previous studies show that Actb knock-out (KO) in mice is lethal while Actg1-KO mice are viable but experience increased health issues. Mice edited to express 𝛾-actin from Actb (Actbc-g) did not experience the same deleterious effects. Both Actg1-KO and Actbc-g mouse embryonic fibroblasts (MEFs) migrate at wild-type rates and maintain constant levels of actin expression through upregulation of other actin isoforms. I examined the phenotypes and actin expression of Actbc-g/Actg1-/- mice. A random cell migration assay was performed to analyze Actbc-g/Actg1 cell migration. I found no significant differences in cell displacement, directionality, or speed between wild-types and Actbc-g/Actg1-/- MEFs. To quantify cytoplasmic actin expression in Actbc-g/Actg1-/- muscle, I attempted a DNase I enrichment. DNase I has a high affinity for monomeric actin and can be used to specifically extract cytoplasmic-actin from tissue that is rich in muscle-specific actin. Protocol optimization steps prevented the completion of this experiment, but I was able to successfully extract 𝛽- and 𝛾-actin from sample tissue.