Patrinostro, Xiaobai2019-08-202019-08-202018-05https://hdl.handle.net/11299/206413University of Minnesota Ph.D. dissertation. May 2018. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: James Ervasti. 1 computer file (PDF); viii, 171 pages.The highly homologous Actb and Actg1 are ubiquitously expressed and are hypothesized to carry out both redundant and unique functions, but studies using genetic knockout and transcript knockdown have yielded conflicting data. To elucidate the cause of this discrepancy, I characterized actin transcript and protein levels, and cellular phenotypes in both gene- and transcript-targeted primary MEFs. Gene targeting of Actb, but not Actg1, led to decreased cell proliferation, decreased cellular ATP levels, and increased serum response factor signaling in primary MEFs. However, SV40 largeT antigen transformed MEFs supported proliferation in the absence of Actb. Consistent with in vivo mouse studies, both gene and transcript targeting approaches demonstrated the loss of Actb is more disruptive to primary MEF function than is the loss of Actg1. Previous mouse models showed that Actb KOs are embryonically lethal while Actg1 KOs are viable. To determine whether the four amino acid differences between the cytoplasmic actins are essential for life, we generated a mouse model where the Actb gene is edited to encode γ-actin protein instead, an allele referred as Actbc-g. In contrast to the lethal phenotype of Actb KOs, homozygous Actbc-g mice were born at Mendelian ratios, do not exhibit early lethality, and Actbc-g MEFs displayed proliferation and random migration rates similar to WT. Nonetheless, Actbc-g mice showed progressive high frequency hearing loss and stereocilia degeneration as previously reported in the hair-cell specific Actb knockout mice. Thus β-actin protein is not universally required for normal cellular function, but is necessary for maintenance of auditory stereocilia.enBeta ActinGamma ActinGenome EngingeeringStereociliaThesis or Dissertation