Abstract Beta- and Gamma-cytoplasmic actin are nearly indistinguishable in their amino acid sequence, but are encoded by different genes that play nonredundant biological roles. The key determinants that drive their functional distinction are unknown. Here, we tested the hypothesis that - and -actin functions are defined by their nucleotide, rather than their amino acid sequence, using targeted editing of the mouse genome. Although previous studies have shown that disruption of -actin gene critically impacts cell migration and mouse embryogenesis, we demonstrate here that generation of a mouse lacking -actin protein by editing -actin gene to encode -actin protein, and vice versa, does not affect cell migration and/or organism survival. Our data suggest that the essential in vivo function of -actin is provided by the gene sequence independent of the encoded protein isoform. We propose that this regulation constitutes a global silent code mechanism that controls the functional diversity of protein isoforms. (eLife, 6:e31661, doi:10.7554/eLife.31661, 2017)