Cell line development is one of the most critical steps in the production of complex recombinant therapeutic proteins such as monoclonal antibodies in mammalian cells. Generation of industrial cell lines is mainly based on the time-consuming and laborious process of selection and screening of a large number of clones. With the increasing demand for therapeutic proteins during the past years, more effort is invested to improve the efficiency of cell line development. In line with this premise, several studies employed expression vector engineering strategies based on incorporation of epigenetic regulatory elements, which can enhance the expression level and stability of the transgenes. Main examples of such elements include ubiquitous chromatin opening elements, scaffold or matrix attachment regions, stabilizing antirepressor elements, and insulators. This work evaluates the utility of the tDNA insulator element for stable expression of an IgG1 monoclonal antibody as well as the enhanced green fluorescent protein (EGFP) reporter gene in Chinese hamster ovary (CHO) cells. Initial analysis of EGFP transfected cells showed improved mean fluorescent intensity in cell pools and single cell clones when tDNA element was included in the expression vector. Our results also indicated up to nine- and sixfold enhancements in antibody titer and specific productivity of clones derived from tDNA containing vectors, respectively. Moreover, improved single cell cloning efficiency was observed for transfectants generated using tDNA harboring expression constructs. Our study clearly shows the beneficial effects of the tDNA insulator on monoclonal antibody expression in CHO cells.