Abstract
Translation is complicated and poorly understood. Translation initiation is most commonly regulated by scientists. Various proteins play important roles in two types of translation initiation; however, different types of translation require different proteins to regulate. 4EBP and eIF4E are crucial in translation. 4EBPs play important roles in regulating translation initiation, however, it is unclear for the importance of the biological role of them for protein synthesis in human cell lines. To investigate the importance of these 4EBP proteins, I will first establish a stable knockout 4EBP1 human cell lines. CRISPR/Cas9-coupled NHEJ repair was utilized to establish stable 4EBP1 knockout HEK293T cell lines. Our results show that CRISPR/Cas9-coupled NHEJ repair is a workable method for knock-in 4EBP genes in mammalian cells. In our experiment, knock- out and knock-in techniques were performed at the same time. To study the impact of different 4EBP genes on the overall protein synthesis, gRNA was designed to knocking out three types of 4EBP genes individually. Moreover, to select the positive monoclonal cell lines, blasticidin resistance marker was inserted into the same location of knockout 4EBP genes with the knock-in helps. Consequently, the stable 4EBP1 knockout human cell lines will provide numerous potential contributions in basic biological research and biomedical research to identify the function of 4EBP1.